U.S. patent application number 14/876376 was filed with the patent office on 2016-05-12 for dosing regimens using anti-il-6 antibodies for the treatment of rheumatoid and psoriatic arthritis.
The applicant listed for this patent is ALDERBIO HOLDINGS LLC. Invention is credited to Jeffrey T.L. Smith.
Application Number | 20160130340 14/876376 |
Document ID | / |
Family ID | 55911706 |
Filed Date | 2016-05-12 |
United States Patent
Application |
20160130340 |
Kind Code |
A1 |
Smith; Jeffrey T.L. |
May 12, 2016 |
DOSING REGIMENS USING ANTI-IL-6 ANTIBODIES FOR THE TREATMENT OF
RHEUMATOID AND PSORIATIC ARTHRITIS
Abstract
The present invention relates to therapeutic methods of using an
antibody, or antigen-binding fragment thereof, which selectively
binds IL-6 for the treatment or prevention of psoriatic arthritis
or rheumatoid arthritis and for managing the side effects and
symptoms of psoriatic or rheumatoid arthritis and therapeutic
compositions for use therein comprising an antibody, or
antigen-binding fragment thereof, which selectively binds IL-6 for
the treatment or prevention of psoriatic or rheumatoid arthritis.
The invention further relates to low dosing therapeutic regimens
for treating inflammatory IL-6 associated diseases, i.e.,
characterized by elevated 11-6 levels such as psoriatic arthritis
or rheumatoid arthritis that provided for reduced adverse side
effects and improved safety. Also the invention further relates to
compositions for use in low dosing therapeutic regimens for
treating inflammatory IL-6 associated diseases, i.e., diseases
characterized by elevated IL-6 levels such as psoriatic arthritis
or rheumatoid arthritis, wherein such compositions may be
administered by self-injection or by a caregiver using an
autoinjector pen and a syringe containing the low dosage of
anti-IL-6 antibody, e.g., 1, 5, 10, 15, 20 or 25 mg.
Inventors: |
Smith; Jeffrey T.L.;
(Bellevue, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALDERBIO HOLDINGS LLC |
LAS VEGAS |
NV |
US |
|
|
Family ID: |
55911706 |
Appl. No.: |
14/876376 |
Filed: |
October 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2014/059543 |
Oct 7, 2014 |
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14876376 |
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62060797 |
Oct 7, 2014 |
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61887666 |
Oct 7, 2013 |
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Current U.S.
Class: |
424/158.1 ;
530/389.2 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/92 20130101; A61K 9/0019 20130101; A61K 45/06 20130101;
C07K 2317/41 20130101; C07K 2317/76 20130101; C07K 2317/565
20130101; C07K 2317/56 20130101; C07K 16/248 20130101; A61K 2300/00
20130101; A61K 2039/545 20130101; C07K 2317/94 20130101; A61K
31/519 20130101; C07K 2317/24 20130101; A61K 31/519 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A61K 39/395 20060101 A61K039/395; A61K 9/00 20060101
A61K009/00; A61K 31/519 20060101 A61K031/519 |
Claims
1. A method for treating or preventing rheumatoid arthritis, or
managing one or more of the symptoms of rheumatoid arthritis
comprising administration of a composition comprising an effective
amount of an anti-IL-6 antibody or antibody fragment thereof to a
subject in need thereof, wherein the anti-IL-6 antibody or antibody
fragment thereof comprises a variable light (V.sub.L) chain
polypeptide comprising a CDR1 sequence of SEQ ID NO:4, a CDR2
sequence of SEQ ID NO:5, and a CDR3 sequence of SEQ ID NO:6, and a
variable heavy (V.sub.H) chain polypeptide comprising a CDR1
sequence of SEQ ID NO:7, a CDR2 sequence of SEQ ID NOs:8 or 120,
and a CDR3 sequence of SEQ ID NO:9, and wherein said anti-IL-6
antibody or antibody fragment thereof is administered every 4 weeks
or monthly at a dosage of at most 1, 5, 10, 15, 20 or 25 mg.
2. The method for treating or preventing rheumatoid arthritis or
managing one or more of the symptoms of rheumatoid arthritis of
claim 15, comprising administration of a composition comprising an
effective amount of an anti-IL-6 antibody or antibody fragment
thereof to a subject in need thereof, wherein the anti-IL-6
antibody or antibody fragment thereof comprises a variable light
(V.sub.L) chain polypeptide comprising the amino acid sequence in
SEQ ID NO:20, 702 or 709, and a variable heavy (V.sub.H) chain
polypeptide comprising the amino acid sequence in SEQ ID NO:18, 19,
657 or 704 and wherein said anti-IL-6 antibody or antibody fragment
thereof is administered every 4 weeks or monthly at a dosage of at
most 1, 5, 10, 15, 20 or 25 mg.
3. The method for treating or preventing rheumatoid arthritis or
managing one or more of the symptoms of rheumatoid arthritis of
claim 1, comprising administration of a composition comprising an
effective amount of an anti-IL-6 antibody or antibody fragment
thereof to a subject in need thereof, wherein the anti-IL-6
antibody or antibody fragment thereof comprises a variable light
(V.sub.L) chain polypeptide comprising the amino acid sequence in
SEQ ID NO:20 or 709, and a variable heavy (V.sub.H) chain
polypeptide comprising the amino acid sequence in SEQ ID NO:18, 19,
or 657, and wherein said anti-IL-6 antibody or antibody fragment
thereof is administered every 4 weeks or monthly at a dosage of at
most 1, 5, 10, 15, 20 or 25 mg.
4. The method for treating or preventing rheumatoid arthritis or
managing one or more of the symptoms of rheumatoid arthritis of
claim 1, comprising administration of a composition comprising an
effective amount of an anti-IL-6 antibody or antibody fragment
thereof to a subject in need thereof, wherein the anti-IL-6
antibody or antibody fragment thereof comprises a light chain
polypeptide comprising the polypeptide having the amino acid
sequence in SEQ ID NO:702 and a heavy chain comprising the
polypeptide having the amino acid sequence of SEQ ID NO:704, and
wherein said anti-IL-6 antibody or antibody fragment thereof is
administered every 4 weeks or monthly at a dosage of at most 1, 5,
10, 15, 20 or 25 mg.
5-35. (canceled)
36. The method of claim 1, wherein said subject has had an
inadequate response to non-steroidal anti-inflammatory drugs
(NSAIDs).
37. The method of claim 1, wherein said subject has had an
inadequate response to non-biologic Disease Modifying
Anti-Rheumatic Drugs (DMARDs).
42. A dosage composition, or syringe or injector pen containing a
single dosage of an anti-IL-6 antibody or antibody fragment which
is for use in treating or preventing rheumatoid arthritis according
to any of the foregoing claims, and wherein said anti-IL-6 antibody
or antibody fragment comprises or consists of CDRs, variable heavy
or light polypeptides or light and heavy polypeptides having the
amino acid sequences as set forth in claim 1, wherein the single
dosage of said anti-IL-6 antibody or antibody fragment contained in
said composition or syringe or injector pen containing same
comprises at most or consists of 1, 5, 10, 15, 20, or 25 mg of said
anti-IL-6 antibody or antibody fragment.
44-47. (canceled)
48. A therapeutic regimen for treating or preventing psoriatic
and/or rheumatoid arthritis or managing the side effects of
psoriatic and/or rheumatoid arthritis in a subject in need thereof,
wherein the therapeutic regimen comprises or consists of
administering a single dosage of an anti-IL-6 antibody or antibody
fragment every 4 weeks or monthly using a syringe or injector pen
which single dosage comprises at most or consists of 1, 5, 10, 15,
20, or 25 mg of an anti-IL-6 antibody or antibody fragment
according to claim 1.
49. The regimen of claim 48, wherein the anti-IL-6 antibody or
antibody fragment comprises the V.sub.L polypeptide of SEQ ID NO:
20 or 709 and V.sub.H polypeptides having the amino acid sequence
of SEQ ID NO: 18, 19 or 657.
49-53. (canceled)
54. The method or regimen of claim 1 or 48, which further includes
the administration of methotrexate.
55. The method or regimen of claim 1 or 48 wherein the treated
subject has developed a resistance or tolerance to
methotrexate.
56. The method or regimen of claim 1 or 48, wherein the treated
subject has previously received methotrexate.
57. The method or regimen of any of the foregoing claims, claim 1
or 48, wherein the treated subject has previously received another
anti-IL-6 antagonist or an anti-TNF biologic.
58. The method or regimen of any of the foregoing claims, wherein
the treated subject has previously received Humira.RTM.,
Remicade.RTM., or Actemra.RTM..
59-65. (canceled)
67. An improved therapeutic regimen for treating rheumatoid
arthritis ("RA") using an anti-IL-6 antibody, wherein the anti-IL-6
antibody comprises the light chain polypeptide of SEQ ID NO:702 and
the heavy chain polypeptide of SEQ ID NO:704, and the anti-IL-6
antibody is administered weekly, every 4 weeks or monthly at a
dosage of at most 1-5 mg or is administered weekly, every 4 weeks
or or monthly at a dosage of at most at most 5-25 mg, and wherein
such regimen provides for greater patient remission at lower
dosages and/or less frequent dosing than current biologics used to
treat RA.
68. The therapeutic regimen of claim 67, which is used to treat an
RA in a patient resistant to methotrexate ("MTX") or to another
biologic.
69. The improved therapeutic regimen of claim 67, wherein the other
biologic is Humira.RTM., Remicade.RTM. or Actemera.RTM..
70. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Ser. No. 62/060,797, filed on Oct. 7, 2014 and PCT
Application entitled "ANTI-IL-6 ANTIBODIES FOR THE TREATMENT OF
PSORIATIC ARTHRITIS" PCT/US2014/059543 filed on Oct. 7, 2014 which
in turn claims priority to U.S. Provisional application No.
61/887,666 filed Oct. 7, 2013, the entire content of which are
hereby incorporated herein by reference in their entirety as though
fully set forth herein.
[0002] This application includes as part of its disclosure a
biological sequence listing text file which is being submitted via
EFS-Web. Said biological sequence listing is contained in the file
named "43272o3005" having a size of 362,137 bytes that was created
Oct. 6, 2015, which is hereby incorporated by reference in its
entirety.
FIELD OF THE SUBJECT TECHNOLOGY
[0003] Anti-IL-6 antibodies and antigen-binding fragments thereof
are used to reduce C-reactive protein ("CRP levels") and
inflammation and in methods and compositions for the treatment and
prevention of psoriatic arthritis (PsA).
BACKGROUND
Interleukin-6 (IL-6)
[0004] Interleukin-6 ("IL-6") is a multifunctional cytokine
involved in numerous biological processes such as the regulation of
the acute inflammatory response, the modulation of specific immune
responses including B- and T-cell differentiation, bone metabolism,
thrombopoiesis, epidermal proliferation, menses, neuronal cell
differentiation, neuroprotection, aging, cancer, and the
inflammatory reaction occurring in Alzheimer's disease. See
Papassotiropoulos, et al. (2001) Neurobiology of Aging 22:
863-871.
[0005] IL-6 is a pleiotropic pro-inflammatory cytokine, which
regulates the acute phase response and the transition from the
innate to the adaptive immune response. IL-6 increases hepatic
synthesis of proteins that are involved in the `acute phase
response` leading to symptoms such as fever, chills, and fatigue.
It stimulates B cell differentiation and secretion of antibodies
and prevents apoptosis of activated B cells. IL-6 activates and
induces proliferation of T cells and in the presence of IL-2,
induces differentiation of mature and immature CD8 T cells into
cytotoxic T cells. IL-6 is also involved in the differentiation of
Th17 cells and IL-17 production and inhibits regulatory T cells
(Treg) differentiation. IL-6 also activates osteoclasts,
synoviocytes, neutrophils, and other hematopoietic cells. Park, et
al. (2007) Bulletin of the NYU Hospital for Joint Diseases 65
(suppl 1): S4-10; Guerre, et al. (1989) J Clin Invest. 83(2):
585-92; Houssiau, et al. (1988) Arthritis Rheum. 31(6): 784-8;
Nishimotor, et al. (2006) Nat Clin Pract Rheumatol. 2(11): 619-26;
Kishimoto (1989) Blood 74(1): 1-10; and Van Snick (1990) Annu Rev
Immunol. 8: 253-78.
[0006] IL-6 is a member of a family of cytokines that promote
cellular responses through a receptor complex consisting of at
least one subunit of the signal-transducing glycoprotein gp130 and
the IL-6 receptor ("IL-6R") (also known as gp80). The IL-6R may
also be present in a soluble form ("sIL-6R"). IL-6 binds to IL-6R,
which then dimerizes the signal-transducing receptor gp130. See
Jones (2005) Immunology 175: 3463-3468.
[0007] In humans, the gene encoding IL-6 is organized in five exons
and four introns, and maps to the short arm of chromosome 7 at
7p21. Translation of IL-6 RNA and post-translational processing
result in the formation of a 21 to 28 kDa protein with 184 amino
acids in its mature form. See Papassotiropoulos, et al. (2001)
Neurobiology of Aging 22:863-871.
[0008] The function of IL-6 is not restricted to the immune
response as it acts in hematopoiesis, thrombopoiesis, osteoclast
formation, elicitation of hepatic acute phase response resulting in
the elevation of C-reactive protein (CRP) and serum amyloid A (SAA)
protein. It is known to be a growth factor for epidermal
keratinocytes, renal mesangial cells, myeloma and plasmacytoma
cells. Grossman, et al. (1989) Prot Natl Acad Sci. 86(16):
6367-6371; Horii, et al. (1989) J Immunol. 143(12): 3949-3955; and
Kawano, et al. (1988) Nature 332: 83-85. IL-6 is produced by a wide
range of cell types including monocytes/macrophages, fibroblasts,
epidermal keratinocytes, vascular endothelial cells, renal
messangial cells, glial cells, condrocytes, T and B-cells and some
tumor cells. Akira, et al. (1990) FASEB J. 4(11): 2860-2867. Except
for tumor cells that constitutively produce IL-6, normal cells do
not express IL-6 unless appropriately stimulated.
[0009] Elevated IL-6 levels have been observed in many types of
cancer, including breast cancer, leukemia, ovarian cancer, prostate
cancer, pancreatic cancer, lymphoma, lung cancer, renal cell
carcinoma, colorectal cancer, and multiple myeloma (e.g., Chopra et
al. (2004) MJAFI 60:45-49; Songur et al. (2004) Tumori 90:196-200;
Blay et al. (1992) Cancer Research 52: 3317-3322; Nikiteas et al.
(2005) World J. Gasterenterol. 11:1639-1643; reviewed in Heikkila
et al. (2008) Eur J Cancer 44:937-945). Clinical studies (reviewed
in Trikha et al. (2003) Clinical Cancer Research 9: 4653-4665) have
shown some improvement in patient outcomes due to administration of
various anti-IL-6 antibodies, particularly in those cancers in
which IL-6 plays a direct role promoting cancer cell proliferation
or survival.
[0010] As noted above, IL-6 stimulates the hepatic acute phase
response, resulting in increased production of CRP and elevated
serum CRP levels. For this reason, C-reactive protein (CRP) has
been reported to comprise a surrogate marker of IL-6 activity.
Thus, elevated IL-6 activity can be detected through measurement of
serum CRP. Conversely, effective suppression of IL-6 activity,
e.g., through administration of a neutralizing anti-IL-6 antibody,
can be detected by the resulting decrease in serum CRP levels.
Although no diagnostic blood tests available for psoriatic
arthritis, elevated CRP levels and erythrocyte sedimentation rate
are known markers of inflammation and may reflect the severity of
inflammation in the joints experienced in psoraitic arthritis.
DermNet NZ by the New Zealand Dermatological Society Incorporated
(2011).
Rheumatoid Arthritis
[0011] Rheumatoid arthritis (RA) is one of the most common forms of
chronic inflammatory arthritis, affecting approximately 1% of the
population worldwide. Women are 2 to 3 times more likely to develop
the disease compared to men, with a peak incidence between the
fourth and sixth decades of life. While RA is recognized clinically
because of the severe inflammation affecting the synovial joints,
it is also a systemic disease with frequent extra-articular
manifestations. The natural history of RA is characterized by joint
destruction, impaired physical function, and poor health-related
quality of life.
[0012] In general, treatment options for RA patients range from
agents that provide symptomatic relief (such as analgesics,
non-steroidal anti-inflammatory drugs [NSAIDs], and
corticosteroids) to disease modifying agents that affect long-term
structural damage. The current approach to RA treatment involves
early intervention and progressive changes of therapy to improve
signs and symptoms, and to prevent long term structural damage.
Patients who are early in their disease course most commonly
initiate treatment with one or more conventional synthetic DMARDs
(eg. MTX). If there is an inadequate response (IR) to signs and
symptoms or physical function with conventional synthetic DMARDs,
these patients are often candidates for biologic therapy, most
commonly anti tumor necrosis factor (anti-TNF) treatments. If
patients do not attain adequate efficacy goals with anti-TNF
agents, they are treated by switching to an alternative biologic
therapy either within the anti-TNF class or with a different
mechanism of action (e.g. co-stimulation blockade, anti-B-cell
therapy, or anti-interleukin-6 [anti-IL-6] therapy).
[0013] Despite ongoing research and therapeutic advances that have
led to significant improvement in patient health, RA remains a
disease with considerable unmet medical need. The biological DMARD
therapies now common in clinical practice perform reasonably well
with respect to ACR criteria for 20% improvement (ACR20) response.
However, their ability to achieve higher levels of efficacy is
quite limited. For example, fewer than half of adult patients with
moderate to severe active RA, who have had an inadequate response
to MTX, achieve ACR criteria for 50% improvement (ACR50) response,
and only approximately 20% of patients achieve ACR criteria for 70%
improvement (ACR70) in recent trials of biologic therapies. More
importantly, few patients achieve sustained levels of low disease
activity or clinical remission. Data from clinical trials suggest
that 5-20% of patients on conventional synthetic DMARD or biologic
monotherapy, and 20-30% of patients on combination DMARD/biologic
therapy achieve low disease activity or remission.
[0014] Low rates of low disease activity are of concern because the
attainment of clinical remission is associated with less long term
structural damage and physical disability. Recent treatment
guidelines from an international task force have highlighted this
need and recommended that control of disease activity is the
therapeutic objective in RA, and have recommended a treat-to-target
approach. As a result, there is a considerable need for new
therapies that can help greater numbers of patients achieve low
disease activity and clinical remission.
[0015] As disclosed infra, Clazakizumab, is a humanized monoclonal
antibody that binds to the IL-6 cytokine which has demonstrated to
be efficacious in RA with an acceptable safety profile and has
shown numerically higher low disease activity and clinical
remission rates as compared to adalimumab in a Phase 2b dose
ranging study.
Psoriatic Arthritis
[0016] Psoriatic arthritis (PsA) is a chronic inflammatory
arthritis that occurs in individuals with psoriasis. It is
estimated that about 1-3% of the general population and
approximately 4.5 million patients in the United States have
psoriasis. Between 10 and 30% of the psoriatic patients develop
arthritis. PsA is a chronic inflammatory disease. The pathogenesis
of PsA is not fully understood. Both genetic predisposition and
environmental triggers are implicated in the deregulation of immune
functions involved in PsA. A number of inflammatory cytokines
including Interferon .gamma. (INF .gamma.), Tumor Necrosis Factor
.alpha. (TNF .alpha.), IL-6, IL-8, IL-12, IL-17, and IL-18 are
involved in the pathogenesis of psoriasis and PsA. Spadaro, et al.
(1996) Clinical and Experimental Rheumatology 14: 413-416; Neuner,
et al. (1991) The Journal of Investigative Dermatology 97(1):
27-33; Arican, et al. (2005) Mediators of Inflammation 5: 273-279;
and Goodman, et al. (2009) The Journal of Immunology.
[0017] Psoriatic arthritis, a seronegative spondyloarthropathy is a
complex disease involving peripheral and axial joints,
periarticular structures (e.g., enthesitis and other soft tissues,
resulting in dactylitis) as well as the skin and nails. Mease
(2006) Bulletin of the NYU Hospital for Joint Diseases. 65(1-2):
25-31. Without appropriate management, the number of joints
affected by PsA and the severity of joint damage increase over
time, which can lead to marked restrictions of the daily activities
and to substantially compromised quality of life. Evidence has
shown that accelerated atherosclerosis, obesity, metabolic syndrome
and cardiovascular disease are associated with active PsA. Other
co-morbidities such as pulmonary fibrosis, uveitis, and, less
commonly, aorta and aortic valve inflammation also contribute to
complexity of PsA. Mease (2005) Expert Opin. Biol. Ther. 5(11):
1491-1504; Mease (2006) Bulletin of the NYU Hospital for Joint
Diseases. 65(1-2): 25-31; and Weger (2010) British Journal of
Pharmacology. 160: 810-820.
[0018] Treatment options are limited for PsA. Saad, et al. (2008) J
Rheumatol. 35(5): 883-890; Kavanuah, et al. (2006) J Rheumatol.
33(7): 1417-142; Gavin, (2010) The Hong Kong Medical Diary. 15(5):
26-27; and Nash (2006) J Rheumatol. 33(7): 1431-1424. Responses to
the traditional disease-modifying anti-rheumatic drugs (DMARDs)
have been suboptimal. Kavanuah, et al. (2006) J Rheumatol. 33(7):
1417-1421 and Soriana, et al. (2006) J Rheumatol. 33(7): 1422-1430.
Anti-tumor necrosis factor (TNF) therapies are efficacious for both
skin and joint diseases but approximately 40% of patients treated
with anti-TNF agents do not show at least minimal improvement and a
large portion of patients do not achieve substantial relief. Mease
(2006) Bulletin of the NYU Hospital for Joint Diseases. 65(1-2):
25-31; Weger (2010) British Journal of Pharmacology. 160: 810-820;
Mease, et al. (2000) The Lancet 356: 385-390. Mease, et al. (2004)
Arthritis and Rheum. 50(7): 2264-2272; Genovese, et al. (2007) J
Rheumatol. 34(5): 1040-1050; Mease, et al. (2005) Arthritis and
Rheum. 52(10: 3279-3289; Mease (2007) Therapeutics and Clinical
Risk Management 3(1): 133-148; Antoni, et al. (2005) Arthritis and
Rheum. 52(4): 1227-1236; Antoni, et al. (2008) J Rheumatol. 35(5):
869-876; and Antoni, et al. (2005) Ann Rhem Dis. 64: 1150-1157. For
example, several effective rheumatoid arthritis therapies do not
provide the relief in PsA, leaving anti-TNF agents as the only
class of approved biologic therapy for PsA. Several agents under
development for PsA had good efficacy for psoriatic skin lesions
but with less optimal joint efficacy. Mease (2006) Bulletin of the
NYU Hospital for Joint Diseases. 65(1-2): 25-31. Weger (2010)
British Journal of Pharmacology. 160: 810-820. Gottlieb, et al.
(2009) The Lancet 373: 633-640.
[0019] More severe arthritis has been treated with drugs called
disease-modifying antirheumatic drugs (DMARDs), such as
Leflunomide, Methotrexate, and Sulfasalazine. New medications that
block an inflammatory protein called tumor necrosis factor (TNF)
are becoming the treatment of choice for psoriatic arthritis
including Adalimumab (Humira), Etanercept (Enbrel), Golimumab
(Simponi), Infliximab (Remicade). Occasionally, very painful joints
may be injected with steroid medications. A.D.A.M. Medical
Encyclopedia (Jun. 29, 2011). However, many patients do not
experience relief from psoriatic arthritis with DMARDs or
non-steroidal anti-inflammatory drugs (NSAIDs). Therefore, there is
still significant unmet need in PsA for therapies that provide
higher levels of efficacy in a greater proportion of patients for
both joints and skin along with the additional attributes of
durability of effect over time, low immunogenicity, a subcutaneous
dosing regimen that may allow for less frequent administration, and
a risk benefit profile that remains acceptable.
[0020] Data suggest that IL-6 plays an important role in the
pathogenesis that leads to the joint inflammation as well as the
characteristic skin lesions associated with psoriasis arthritis.
Spadaro, et al. (1996) Clinical and Experimental Rheumatology 14:
413-416. Neuner, et al. (1991) The Journal of Investigative
Dermatology 97(1): 27-33. Arican, et al. (2005) Mediators of
Inflammation 5: 273-279. Goodman, et al. (2009) The Journal of
Immunology. Grossman, et al. (1989) Medical Sciences, Proc. Natl.
Acad. Sci. USA. 86: 6367-6371. Circulating IL-6 levels were
significantly higher in PsA patients and correlated highly with ESR
and CRP. IL-6 levels also strongly correlated with disease
activities such as the number of painful and swollen joints,
physician's assessments as well as psoriasis area and severity
index (PASI). Spadaro, et al. (1996) Clinical and Experimental
Rheumatology 14: 413-416. The histopathology of psoriatic skin
lesions is characterized by epidermal hyperplasia and inflammation.
Studies have shown IL-6 mRNA and protein levels are elevated in
psoriatic plaques. IL-6 was also shown to stimulate keratinocyte
proliferation in vitro and contribute to the plaque formation. IL-6
signaling in psoriasis was also shown to prevent immune suppression
by regulatory T cells. Neuner, et al. (1991) The Journal of
Investigative Dermatology 97(1): 27-33. Arican, et al. (2005)
Mediators of Inflammation 5: 273-279. Goodman, et al. (2009) The
Journal of Immunology. Grossman, et al. (1989) Medical Sciences,
Proc. Natl. Acad. Sci. USA. 86: 6367-6371. Alenius, et al. (2009)
Clinical and Experimental Rheumatology 27: 120-123. Therefore,
blocking IL-6 may provide therapeutic benefits in PsA.
[0021] Therefore, there is a strong need in the art for improved
methods of treating and preventing psoriatic arthritis.
SUMMARY
[0022] The present technology provides compositions comprising
humanized monoclonal antibodies that selectively bind IL-6 and
methods of treating psoriatic arthritis. In one embodiment,
anti-IL-6 antibodies (e.g., ALD518 antibodies) are used in methods
for the treatment of psoriatic arthritis. In this embodiment of the
subject technology anti-IL-6 antibody or antibody fragment are
administered prophylactically to patients at significant risk of
developing psoriatic arthritis. The subject technology also
provides for humanized monoclonal anti-IL-6 antibodies which are
used in the treatment of psoriatic arthritis. The present subject
technology further includes the prevention or treatment of
inflammatory conditions by administration of anti-IL-6 antibodies
according to the subject technology.
[0023] The subject technology provides a method of treating or
preventing psoriatic arthritis comprising administration of a
composition comprising an effective amount of an Ab1, Ab2, Ab3,
Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15,
Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,
Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36
antibody, or an antigen-binding fragment thereof, to a subject in
need thereof, wherein the antibody, or antigen-binding fragment
thereof, specifically binds to IL-6.
[0024] The subject technology also provides a method of treating
psoriatic arthritis comprising administration of a composition
comprising an effective amount of an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6,
Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17,
Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28,
Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36 antibody, or an
antigen-binding fragment thereof, to a subject in need thereof,
wherein the antibody, or antigen-binding fragment thereof,
specifically binds to IL-6.
[0025] The subject technology further provides a method of
preventing psoriatic arthritis comprising administration of a
composition comprising an effective amount of an Ab1, Ab2, Ab3,
Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15,
Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,
Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36
antibody, or an antigen-binding fragment thereof, to a subject in
need thereof, wherein the antibody, or antigen-binding fragment
thereof, specifically binds to IL-6.
[0026] The subject technology provides a composition for the
treatment or prevention of psoriatic arthritis comprising an
effective amount of an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,
Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20,
Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31,
Ab32, Ab33, Ab34, Ab35, or Ab36 antibody, or an antigen-binding
fragment thereof, to a subject in need thereof, wherein the
antibody, or antigen-binding fragment thereof, specifically binds
to IL-6.
[0027] The subject technology also provides a composition for the
treatment of psoriatic arthritis comprising an effective amount of
an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12,
Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23,
Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34,
Ab35, or Ab36 antibody, or an antigen-binding fragment thereof, to
a subject in need thereof, wherein the antibody, or antigen-binding
fragment thereof, specifically binds to IL-6.
[0028] The subject technology further provides a composition for
the prevention of psoriatic arthritis comprising an effective
amount of an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10,
Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21,
Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32,
Ab33, Ab34, Ab35, or Ab36 antibody, or an antigen-binding fragment
thereof, to a subject in need thereof, wherein the antibody, or
antigen-binding fragment thereof, specifically binds to IL-6.
[0029] The subject technology provides a composition comprising an
effective amount of an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,
Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20,
Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31,
Ab32, Ab33, Ab34, Ab35, or Ab36 antibody, or an antigen-binding
fragment thereof, to a subject in need thereof, wherein the
antibody, or antigen-binding fragment thereof, specifically binds
to IL-6.
[0030] The subject technology also provides for a pharmaceutical
composition comprising an effective amount of an Ab1, Ab2, Ab3,
Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15,
Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,
Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36
antibody, or an antigen-binding fragment thereof, to a subject in
need thereof, wherein the antibody, or antigen-binding fragment
thereof, specifically binds to IL-6.
[0031] The subject technology provides for the use of a composition
comprising an effective amount of an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6,
Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17,
Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28,
Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36 antibody, or an
antigen-binding fragment thereof, to a subject in need thereof,
wherein the antibody, or antigen-binding fragment thereof,
specifically binds to IL-6, for the manufacture of a medicament for
the treatment or prevention of psoriatic arthritis. In a further
embodiment, said composition may be formulated for subcutaneous
administration.
[0032] The subject technology also provides for the use of a
composition comprising an effective amount of an Ab1, Ab2, Ab3,
Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15,
Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,
Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36
antibody, or an antigen-binding fragment thereof, to a subject in
need thereof, wherein the antibody, or antigen-binding fragment
thereof, specifically binds to IL-6, for the manufacture of a
medicament for the treatment of psoriatic arthritis. In a further
embodiment, said composition may be formulated for subcutaneous
administration.
[0033] The subject technology provides for the use of a composition
comprising an effective amount of an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6,
Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17,
Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28,
Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36 antibody, or an
antigen-binding fragment thereof, to a subject in need thereof,
wherein the antibody, or antigen-binding fragment thereof,
specifically binds to IL-6, for the manufacture of a medicament for
the prevention of psoriatic arthritis. In a further embodiment,
said composition may be formulated for subcutaneous
administration.
[0034] In one embodiment, the antibody includes at least one light
chain amino acid sequence with at least about 50% identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 2, 20, 21, 37, 53, 69, 85, 101, 119, 122, 138, 154, 170, 186,
202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394,
410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 647, 648,
649, 650, 651, 655, 660, 666, 667, 671, 675, 679, 683, 687, 693,
699, 702, 706, and 709. In another embodiment, the antibody may
comprise at least one light chain of nucleic acid sequences with at
least 50% identity to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 10, 29, 45, 61, 77, 93, 109, 130,
146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322, 338,
354, 370, 386, 402, 418, 434, 450, 466, 482, 498, 514, 530, 546,
562, 578, 662, 669, 673, 677, 681, 685, 689, 698, 701, 705, 720,
721, 722, and 723, wherein said nucleic acid sequence encodes said
light chain.
[0035] In one embodiment, the antibody includes at least one heavy
chain amino acid sequence with at least about 50% identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 3, 18, 19, 22, 38, 54, 70, 86, 102, 117, 118, 123, 139, 155,
171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363,
379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555, 571,
652, 653, 654, 655, 656, 657, 658, 661, 664, 665, 668, 672, 676,
680, 684, 688, 691, 692, 704, and 708. In another embodiment, the
antibody includes at least one heavy chain nucleic acid sequences
with at least 50% identity to a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 11, 30, 46, 62, 78, 94, 110,
131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, 307, 323,
339, 355, 371, 387, 403, 419, 435, 451, 467, 483, 499, 515, 531,
547, 563, 579, 663, 670, 674, 678, 682, 686, 690, 700, 703, 707,
724, and 725, wherein said nucleic acid sequence encodes said heavy
chain.
[0036] In one embodiment, the antibody includes at least one CDR
amino acid sequence with at least about 50% identity to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 4,
7, 23, 26, 39, 42, 55, 58, 71, 74, 87, 90, 103, 106, 124, 127, 140,
143, 156, 159, 172, 175, 188, 191, 204, 207, 220, 223, 236, 239,
252, 255, 268, 271, 284, 287, 300, 303, 316, 319, 332, 335, 348,
351, 364, 367, 380, 383, 396, 399, 412, 415, 428, 431, 444, 447,
460, 463, 476, 479, 492, 495, 508, 511, 524, 527, 540, 543, 556,
559, 572, 575, 710, 711, 712, 716, 5, 8, 24, 27, 40, 43, 56, 59,
72, 75, 88, 91, 104, 107, 120, 121, 125, 128, 141, 144, 157, 160,
173, 176, 189, 192, 205, 208, 221, 224, 237, 240, 253, 256, 269,
272, 285, 288, 301, 304, 317, 320, 333, 336, 349, 352, 365, 368,
381, 384, 397, 400, 413, 416, 429, 432, 445, 448, 461, 464, 477,
480, 493, 496, 509, 512, 525, 528, 541, 544, 557, 560, 573, 576,
659, 713, 714, 715, 717, 718, 6, 9, 25, 28, 41, 44, 57, 60, 73, 76,
89, 92, 105, 108, 126, 129, 142, 145, 158, 161, 174, 177, 190, 193,
206, 209, 222, 225, 238, 241, 254, 257, 270, 273, 286, 289, 302,
305, 318, 321, 334, 337, 350, 353, 366, 369, 382, 385, 398, 401,
414, 417, 430, 433, 446, 449, 462, 465, 478, 481, 494, 497, 510,
513, 526, 529, 542, 545, 558, 561, 574, and 577. In one embodiment,
the antibody includes at least one CDR nucleic acid sequences with
at least 50% identity to a nucleic acid sequence selected from the
group consisting of SEQ ID NOs: 12, 15, 31, 34, 47, 50, 63, 66, 79,
82, 95, 98, 111, 114, 132, 135, 148, 151, 164, 167, 180, 183, 196,
199, 212, 215, 228, 231, 244, 247, 260, 263, 276, 279, 292, 295,
308, 311, 324, 327, 340, 343, 356, 359, 372, 375, 388, 391, 404,
407, 420, 423, 436, 439, 452, 455, 468, 471, 484, 487, 500, 503,
516, 519, 532, 535, 548, 551, 564, 567, 580, 583, 694, 13, 16, 32,
35, 48, 51, 64, 67, 80, 83, 96, 99, 112, 115, 133, 136, 149, 152,
165, 168, 181, 184, 197, 200, 213, 216, 229, 232, 245, 248, 261,
264, 277, 280, 293, 296, 309, 312, 325, 328, 341, 344, 357, 360,
373, 376, 389, 392, 405, 408, 421, 424, 437, 440, 453, 456, 469,
472, 485, 488, 501, 504, 517, 520, 533, 536, 549, 552, 565, 568,
581, 584, 696, 14, 17, 33, 36, 49, 52, 65, 68, 81, 84, 97, 100,
113, 116, 134, 137, 150, 153, 166, 169, 182, 185, 198, 201, 214,
217, 230, 233, 246, 249, 262, 265, 278, 281, 294, 297, 310, 313,
326, 329, 342, 345, 358, 361, 374, 377, 390, 393, 406, 409, 422,
425, 438, 441, 454, 457, 470, 473, 486, 489, 502, 505, 518, 521,
534, 537, 550, 553, 566, 569, 582, 585, 695, and 697, wherein said
nucleic acid sequence encodes said CDR sequence.
[0037] In another embodiment, the antibody or antigen-binding
fragment thereof includes at least one light chain CDR amino acid
sequence with at least about 50% identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 4, 23, 39, 55,
71, 74, 87, 103, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268,
284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476,
492, 508, 524, 540, 556, 572, 710, 711, 712, 5, 6, 24, 40, 56, 72,
88, 104, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285,
301, 317, 333, 349, 365, 381, 397, 413, 429, 445, 461, 477, 493,
509, 525, 541, 557, 573, 713, 714, 715, 718, 25, 41, 57, 73, 89,
105, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302,
318, 334, 350, 366, 382, 398, 414, 430, 446, 462, 478, 494, 510,
526, 542, 558, and 574. In another embodiment, the antibody or
antigen-binding fragment thereof includes at least one light chain
CDR1 amino acid sequence with at least about 50% identity to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 4, 23, 39, 55, 71, 74, 87, 103, 124, 140, 156, 172, 188, 204,
220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412,
428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 710, 711, and
712. In another embodiment, the antibody or antigen-binding
fragment thereof includes at least one light chain CDR2 amino acid
sequence with at least about 50% identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 5, 24, 40, 56,
72, 88, 104, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285,
301, 317, 333, 349, 365, 381, 397, 413, 429, 445, 461,7 477, 493,
509, 525, 541, 557, 573, 713, 714, 715, and 718. In another
embodiment, the antibody or antigen-binding fragment thereof
includes at least one light chain CDR3 amino acid sequence with at
least about 50% identity to an amino acid sequence selected from
the group consisting of SEQ ID NOs: 6, 25, 41, 57, 73, 89, 105,
126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318,
334, 350, 366, 382, 398, 414, 430, 446, 462, 478, 494, 510, 526,
542, 558, and 574. In another embodiment, the antibody or
antigen-binding fragment thereof includes at least two light chain
CDR polypeptides. In another embodiment, the antibody or
antigen-binding fragment thereof may comprise three light chain CDR
polypeptides.
[0038] In another embodiment, the antibody or antigen-binding
fragment thereof includes at least one heavy chain CDR amino acid
sequence with at least about 50% identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7, 26, 42, 58,
74, 90, 106, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287,
303, 319, 335, 351, 367, 383, 399, 415, 431, 447, 463, 479, 495,
511, 527, 543, 559, 575, 716, 8, 27, 43, 59, 75, 91, 107, 120, 121,
128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320,
336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496, 512, 528,
544, 560, 576, 659, 717, 718, 9, 28, 44, 60, 76, 92, 108, 129, 145,
161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353,
369, 385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561,
and 577. In a further embodiment, the antibody or antigen-binding
fragment thereof includes at least one heavy chain CDR1 amino acid
sequence with at least about 50% identity to an amino acid sequence
selected from the group consisting of SEQ ID NOs: 7, 26, 42, 58,
74, 90, 106, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287,
303, 319, 335, 351, 367, 383, 399, 415, 431, 447, 463, 479, 495,
511, 527, 543, 559, 575, and 716. In a further embodiment, the
antibody or antigen-binding fragment thereof includes at least one
heavy chain CDR2 amino acid sequence with at least about 50%
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 8, 27, 43, 59, 75, 91, 107, 120, 121,
128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320,
336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496, 512, 528,
544, 560, 576, 659, 717, and 718. In a further embodiment, the
antibody or antigen-binding fragment thereof includes at least one
heavy chain CDR3 amino acid sequence with at least about 50%
identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 9, 28, 44, 60, 76, 92, 108, 129, 145,
161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353,
369, 385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561,
and 577. In a further embodiment, the antibody or antigen-binding
fragment thereof includes at least two heavy chain CDR amino acid
sequences. In a further embodiment, the antibody or antigen-binding
fragment thereof includes three heavy chain CDR amino acid
sequences.
[0039] In one embodiment, the light chain of said antibody is
selected from the amino acid sequences of light chains listed in
TABLE 1. In one embodiment, the light chain of said antibody is
selected from the amino acid sequences of heavy chains listed in
TABLE 1. In one embodiment, at least one CDR of said antibody is
selected from the amino acid sequences of CDRs listed in TABLE 1.
In another embodiment, the light chain has at least 80% identity to
an amino acid sequence listed in TABLE 1. In another embodiment,
the light chain has at least 90% identity to an amino acid sequence
listed in TABLE 1. In another embodiment, the light chain includes
an amino acid sequence listed in TABLE 1. In further embodiment,
the heavy chain has at least 80% identity to an amino acid sequence
listed in TABLE 1. In further embodiment, the heavy chain has at
least 90% identity to an amino acid sequence listed in TABLE 1. In
further embodiment, the heavy chain includes an amino acid sequence
listed in TABLE 1. In a still further embodiment, the CDR sequence
of the antibody has at least 80% identity to an amino acid sequence
listed in TABLE 1. In a still further embodiment, the CDR sequence
of the antibody has at least 90% identity to an amino acid sequence
listed in TABLE 1. In a still further embodiment, the CDR sequence
of the antibody includes an amino acid sequence listed in TABLE
1.
[0040] In one embodiment, the antibody or antigen-binding fragment
thereof includes at least one of the CDRs contained in the V.sub.H
polypeptide sequences comprising: SEQ ID NO: 3, 18, 19, 22, 38, 54,
70, 86, 102, 117, 118, 123, 139, 155, 171, 187, 203, 219, 235, 251,
267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459,
475, 491, 507, 523, 539, 555, 571, 652, 656, 657, 658, 661, 664,
665, 668, 672, 676, 680, 684, 688, 691, 692, 704, or 708 and/or at
least one of the CDRs contained in the V.sub.L polypeptide sequence
consisting of: 2, 20, 21, 37, 53, 69, 85, 101, 119, 122, 138, 154,
170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362,
378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554, 570,
647, 651, 660, 666, 667, 671, 675, 679, 683, 687, 693, 699, 702,
706, or 709.
[0041] In one embodiment, the antibody is an Ab1 antibody. In one
embodiment, the antibody includes a light chain comprising the
amino acid sequence of SEQ ID NO: 2, 20, 647, 648, 649, 650, 651,
660, 666, 699, 702, 706, or 709. In one embodiment, the antibody
includes a humanized light chain comprising the amino acid sequence
of SEQ ID NO: 648, 649, and 650. In one embodiment, the antibody
includes at least one light chain CDR comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 4, 5, 6,
710, 711, 712, 713, 714, and 715. In one embodiment, the antibody
includes at least one humanized light chain CDR comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO: 710, 711, 712, 713, 714, and 715. In another embodiment, the
antibody includes a heavy chain comprising the amino acid sequence
of SEQ ID NO: 3, 18, 19, 652, 653, 654, 655, 656, 657, 658, 661,
664, 665, 704, 708. In another embodiment, the antibody includes a
humanized heavy chain comprising the amino acid sequence of SEQ ID
NO: 653, 654, and 655. In another embodiment, the antibody includes
at least one heavy chain CDR comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 7, 9, 74, 716, 8,
120, 659, 717, and 718. In another embodiment, the antibody
includes at least one humanized heavy chain CDR comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO: 74, 716, 717, and 718.
[0042] In one embodiment, the antibody or antigen-binding fragment
thereof includes a Fab, Fab', F(ab').sub.2, Fv, scFv, IgNAR, SMIP,
camelbody, or nanobody. In one embodiment, the antibody or
antigen-binding fragment thereof may have an in vivo half-life of
at least about 30 days in a healthy human subject. In one
embodiment, the antibody or antigen-binding fragment thereof may
have a binding affinity (Kd) for IL-6 of less than about 50
picomolar, or a rate of dissociation (K.sub.off) from IL-6 of less
than or equal to 10.sup.-4 S.sup.-1. In one embodiment, the
antibody or antigen-binding fragment thereof may specifically binds
to the same linear or conformational epitope(s) and/or competes for
binding to the same linear or conformational epitope(s) on an
intact human IL-6 polypeptide or fragment thereof as an anti-IL-6
antibody comprising the polypeptides of SEQ ID NO: 702 and SEQ ID
NO: 704 or the polypeptides of SEQ ID NO: 2 and SEQ ID NO: 3. In
one embodiment, the binding to the same linear or conformational
epitope(s) and/or competition for binding to the same linear or
conformational epitope(s) on an intact human IL-6 polypeptide or
fragment thereof is ascertained by epitopic mapping using
overlapping linear peptide fragments which span the full length of
the native human IL-6 polypeptide and includes at least one
residues comprised in IL-6 fragments selected from those
respectively encompassing amino acid residues 37-51, amino acid
residues 70-84, amino acid residues 169-183, amino acid residues
31-45 and/or amino acid residues 58-72 of SEQ ID NO: 1.
[0043] In one embodiment, the antibody, or antigen-binding fragment
thereof, may be aglycosylated. In one embodiment, the antibody, or
antigen-binding fragment thereof, contains an Fc region that has
been modified to alter effector function, half-life, proteolysis,
and/or glycosylation. In one embodiment, the antibody, or
antigen-binding fragment thereof, is a human, humanized, single
chain, or chimeric antibody. In one embodiment, the antibody, or
antigen-binding fragment thereof, includes a Fab, Fab',
F(ab').sub.2, Fv, or scFv. In one embodiment, the antibody, or
antigen-binding fragment thereof, further comprises a human
F.sub.c. In another embodiment, the F.sub.c is derived from IgG1,
IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11,
IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, or IgG19.
[0044] In one embodiment, the composition includes at least about
25, 80, 100, 160, 200, or 320 mg. In one embodiment, the effective
amount is between about 0.1 and 100 mg/kg of body weight of the
subject. In one embodiment, the subject is administered at least 1,
2, 3, 4, 5, 7, 8, 9 or 10 doses. In one embodiment, composition is
administered every 4 weeks. In one embodiment, the subject is
administered 25 mg every 4 weeks. In one embodiment, the subject is
administered 80 mg every 4 weeks. In one embodiment, the subject is
administered 100 mg every 4 weeks. In one embodiment, the subject
is administered 160 mg every 4 weeks. In one embodiment, the
subject is administered 200 mg every 4 weeks. In one embodiment,
the subject is administered 320 mg every 4 weeks. In another
embodiment, the composition is administered every 4 weeks for at
least 16 weeks. In another embodiment, the composition is
administered every 4 weeks for at least 24 weeks.
[0045] In one embodiment, the patient to whom the methods and
compositions of the subject technology are applied may have an
elevated C-reactive protein ("CRP"). In one embodiment, the patient
may have an elevated IL-6 serum level. In one embodiment, the
patient may have an elevated IL-6 level in the joints. In one
embodiment, the patient may have had an inadequate response to
non-steroidal anti-inflammatory drugs (NSAIDs). In one embodiment,
the patient may have had an inadequate response to non-biologic
Disease Modifying Anti-Rheumatic Drugs (DMARDs).
[0046] In one embodiment, the antibody, or antigen-binding fragment
thereof, inhibits at least one activity associated with IL-6. In
another embodiment, the at least one activity associated with IL-6
is an in vitro activity comprising stimulation of proliferation of
T1165 cells; binding of IL-6 to IL-6R; activation (dimerization) of
the gp130 signal-transducing glycoprotein; formation of
IL-6/IL-6R/130 multimers; stimulation of haptoglobin production by
HepG2 cells modified to express human IL-6 receptor; or any
combination thereof. In one embodiment, prior to administration of
the antibody, or antigen-binding fragment thereof, the subject has
exhibited or is at risk for developing at least one of the
following symptoms: elevated serum C-reactive protein ("CRP");
elevated erythrocyte sedimentation rate; or a combination
thereof.
[0047] In one embodiment, the antibody or antigen-binding fragment
is directly or indirectly coupled to a detectable label, cytotoxic
agent, therapeutic agent, or an immunosuppressive agent. In one
embodiment, the detectable label includes a fluorescent dye,
bioluminescent material, radioactive material, chemiluminescent
moietie, streptavidin, avidin, biotin, radioactive material,
enzyme, substrate, horseradish peroxidase, acetylcholinesterase,
alkaline phosphatase, .beta.-galactosidase, luciferase, rhodamine,
fluorescein, fluorescein isothiocyanate, umbelliferone,
dichlorotriazinylamine, phycoerythrin, dansyl chloride, luminol,
luciferin, aequorin, Iodine 125 (.sup.125I), Carbon 14 (.sup.14C),
Sulfur 35 (.sup.35S), Tritium (.sup.3H), Phosphorus 32 (.sup.32P),
or any combination thereof.
[0048] In one embodiment, the antibody or antigen-binding fragment
is co-administered with another therapeutic agent selected from the
group consisting of analgesics, antibiotics, anti-cachexia agents,
anti-coagulants, anti-cytokine agents, antiemetic agents,
anti-fatigue agent, anti-fever agent, anti-inflammatory agents,
anti-nausea agents, antipyretics, antiviral agents, anti-weakness
agent, chemotherapy agents, cytokine antagonist, cytokines,
cytotoxic agents, gene therapy agents, growth factor, IL-6
antagonists, immunosuppressive agents, statins, and any combination
thereof. In one embodiment, the cytokine antagonist is an
antagonist of a factor comprising tumor necrosis factor-alpha,
interferon gamma, interleukin 1 alpha, interleukin 1 beta,
interleukin 6, or any combination thereof. In one embodiment, the
cytokine antagonist is an antagonist of TNF-.alpha., IL-1.alpha.,
IL-1.beta., IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18,
IFN-.alpha., IFN-.gamma., BAFF, CXCL13, IP-10, leukemia-inhibitory
factor, or a combination thereof. In one embodiment, the growth
factor is VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF),
Hepcidin, or any combination thereof. In one embodiment, the IL-6
antagonist includes an anti-IL-6 antibodies or antigen-binding
fragments thereof, antisense nucleic acids, polypeptides, small
molecules, or any combination thereof.
[0049] In another embodiment, the antisense nucleic acid includes
at least approximately 10 nucleotides of a sequence encoding IL-6,
IL-6 receptor alpha, gp130, p38 MAP kinase, JAK1, JAK2, JAK3, or
SYK. In another embodiment, the antisense nucleic acid includes
DNA, RNA, peptide nucleic acid, locked nucleic acid, morpholino
(phosphorodiamidate morpholino oligo), glycerol nucleic acid,
threose nucleic acid, or any combination thereof. In another
embodiment, the IL-6 antagonist polypeptide includes a fragment of
a polypeptide having a sequence selected from the group consisting
IL-6, IL-6 receptor alpha, gp130, p38 MAP kinase, JAK1, JAK2, JAK3,
and SYK. In another embodiment, the fragment is at least about 40
amino acids in length. In another embodiment, the IL-6 antagonist
includes a soluble IL-6, IL-6 receptor alpha, gp130, p38 MAP
kinase, JAK1, JAK2, JAK3, SYK, or any combination thereof. In
another embodiment, the IL-6 antagonist may be coupled to a
half-life increasing moiety.
[0050] In one embodiment, the antibody or antigen-binding fragment
thereof is administered to the subject in the form of at least one
nucleic acids that encode the antibody. In one embodiment, the
light chain of said antibody or antigen-binding fragment thereof is
encoded by at least one of the following nucleic acid sequences of
SEQ ID NOs: 10, 29, 45, 61, 77, 93, 109, 130, 146, 162, 178, 194,
210, 226, 242, 258, 274, 290, 306, 322, 338, 354, 370, 386, 402,
418, 434, 450, 466, 482, 498, 514, 530, 546, 562, 578, 662, 669,
673, 677, 681, 685, 689, 698, 701, 705, 720, 721, 722, or 723. In
another embodiment, the heavy chain of said antibody or
antigen-binding fragment thereof is encoded by at least one of the
following nucleic acid sequences of SEQ ID NOs: 11, 30, 46, 62, 78,
94, 110, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291,
307, 323, 339, 355, 371, 387, 403, 419, 435, 451, 467, 483, 499,
515, 531, 547, 563, 579, 663, 670, 674, 678, 682, 686, 690, 700,
703, 707, 724, or 725. In another embodiment, at least one of the
CDRs of said antibody or antigen-binding fragment thereof is
encoded by at least one of the following nucleic acid sequences of
SEQ ID NOs: 12, 15, 31, 34, 47, 50, 63, 66, 79, 82, 95, 98, 111,
114, 132, 135, 148, 151, 164, 167, 180, 183, 196, 199, 212, 215,
228, 231, 244, 247, 260, 263, 276, 279, 292, 295, 308, 311, 324,
327, 340, 343, 356, 359, 372, 375, 388, 391, 404, 407, 420, 423,
436, 439, 452, 455, 468, 471, 484, 487, 500, 503, 516, 519, 532,
535, 548, 551, 564, 567, 580, 583, 694, 13, 16, 32, 35, 48, 51, 64,
67, 80, 83, 96, 99, 112, 115, 133, 136, 149, 152, 165, 168, 181,
184, 197, 200, 213, 216, 229, 232, 245, 248, 261, 264, 277, 280,
293, 296, 309, 312, 325, 328, 341, 344, 357, 360, 373, 376, 389,
392, 405, 408, 421, 424, 437, 440, 453, 456, 469, 472, 485, 488,
501, 504, 517, 520, 533, 536, 549, 552, 565, 568, 581, 584, 696,
14, 17, 33, 36, 49, 52, 65, 68, 81, 84, 97, 100, 113, 116, 134,
137, 150, 153, 166, 169, 182, 185, 198, 201, 214, 217, 230, 233,
246, 249, 262, 265, 278, 281, 294, 297, 310, 313, 326, 329, 342,
345, 358, 361, 374, 377, 390, 393, 406, 409, 422, 425, 438, 441,
454, 457, 470, 473, 486, 489, 502, 505, 518, 521, 534, 537, 550,
553, 566, 569, 582, 585, 695, or 697. In another embodiment, at
least one nucleic acids includes the heavy and light chain
polynucleotide sequences of SEQ ID NO: 723 and SEQ ID NO: 700; SEQ
ID NO: 701 and SEQ ID NO: 703; SEQ ID NO: 705 and SEQ ID NO: 707;
SEQ ID NO: 720 and SEQ ID NO: 724; and SEQ ID NO: 10 and SEQ ID NO:
11.
[0051] In one embodiment, the composition is administered
subcutaneously. In another embodiment, the composition is a
pharmaceutical composition. In a further embodiment, the
composition is formulated for subcutaneous administration.
[0052] In one embodiment, the antibody or antigen-binding fragment
thereof is asialated. In one embodiment, the antibody or
antigen-binding fragment thereof is humanized. In one embodiment,
the antibody or antigen-binding fragment thereof has a half-life of
at least about 30 days. In one embodiment, the antibody or
antigen-binding fragment thereof includes the humanized variable
light sequence of amino acid sequence of SEQ ID NO: 20. In one
embodiment, the antibody or antigen-binding fragment thereof
includes humanized variable heavy sequence of amino acid sequence
of SEQ ID NO: 19. In another embodiment, the antibody or
antigen-binding fragment thereof includes at least one light chain
CDRs as set forth in the amino acid sequence of SEQ ID NOs: 4, 5,
or 6. In another embodiment, the antibody or antigen-binding
fragment thereof includes at least one heavy chain CDRs as set
forth in the amino acid sequence of SEQ ID NOs: 7, 120, 8, or 9. In
further embodiment, the antibody or antigen-binding fragment
thereof is an asialated, humanized anti-IL-6 monoclonal antibody
with a half-life of .about.30 days comprising the humanized
variable light and heavy sequences as set forth in SEQ ID NO: 20
and 19 or 702 and 704, respectively.
[0053] One embodiment encompasses specific humanized antibodies and
fragments and variants thereof for treatment or prevention of
psoriatic arthritis capable of binding to IL-6 and/or the
IL-6/IL-6R complex. These antibodies bind to soluble IL-6 or cell
surface expressed IL-6. Also, these antibodies inhibit the
formation or the biological effects of at least one of IL-6,
IL-6/IL-6R complexes, IL-6/IL-6R/gp130 complexes and/or multimers
of IL-6/IL-6R/gp130. The present subject technology relates to
novel therapies and therapeutic protocols using anti-IL-6
antibodies, preferably those described herein.
[0054] In a preferred embodiment this is effected by the
administration of the antibodies described herein, comprising the
sequences of the V.sub.H, V.sub.L and CDR polypeptides described in
Table 1, or humanized or chimeric or single chain versions thereof
containing at least one of the CDRs of the exemplified anti-IL-6
antibody sequences and the polynucleotides encoding them.
Preferably these antibodies will be aglycosylated. In more specific
embodiments of the subject technology these antibodies will block
gp130 activation and/or possess binding affinities (Kds) less than
50 picomolar and/or K.sub.off values less than or equal to
10.sup.-4 S.sup.-1.
[0055] The subject technology also contemplates methods of making
said humanized anti-IL-6 or anti-IL-6/IL-6R complex antibodies and
binding fragments and variants thereof. In one embodiment, binding
fragments include, but are not limited to, Fab, Fab', F(ab').sub.2,
Fv and scFv fragments.
[0056] In one embodiment, the anti-IL-6 antibodies block the
effects of IL-6. In another embodiment, the anti-IL-6 antibody is a
humanized monoclonal antibody that binds to free human IL-6 and
soluble IL-6R/IL-6 complex with an affinity of at least about 4 pM.
In another embodiment, the anti-IL-6 antibody, has a serum
half-life about at least 30 days. In another embodiment, the
anti-IL-6 antibody is based on a consensus human IgG1 kappa
framework that had asparagines modified to alanine to eliminate
N-glycosylation sites.
[0057] In another embodiment, the antibodies and humanized versions
are derived from rabbit immune cells (B lymphocytes) and are
selected based on their homology (sequence identity) to human germ
line sequences. These antibodies may require minimal or no sequence
modifications, thereby facilitating retention of functional
properties after humanization. In exemplary embodiments, the
humanized antibodies include human frameworks which are highly
homologous (possess high level of sequence identity) to that of a
parent (e.g. rabbit) antibody.
[0058] In an embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof specifically bind
to the same linear or conformational epitopes on an intact IL-6
polypeptide or fragment thereof which may include at least
fragments selected from those encompassing amino acid residues
37-51, amino acid residues 70-84, amino acid residues 169-183,
amino acid residues 31-45 and/or amino acid residues 58-72.
[0059] In a preferred exemplary embodiment, the anti-IL-6 antibody
comprises at least one of the CDRs in listed in Table 1. In a more
preferred embodiment the anti-IL-6 antibody comprises the variable
heavy and light chain sequences in SEQ ID NO: 657 and SEQ ID NO:
709, or variants thereof.
[0060] In a preferred embodiment the humanized anti-IL-6 antibody
comprises the variable heavy and variable light chain sequences
respectively set forth in SEQ ID NO: 657 and SEQ ID NO: 709,
respectively, and preferably further comprising the heavy chain and
light chain constant regions respectively set forth in SEQ ID NO:
588 and SEQ ID NO: 586, and variants thereof comprising at least
one amino acid substitutions or deletions that do not substantially
affect IL-6 binding and/or desired effector function. This
embodiment also contemplates polynucleotides comprising, or
alternatively consisting of, at least one of the nucleic acids
encoding the variable heavy chain (SEQ ID NO: 700) and variable
light chain (SEQ ID NO: 723) sequences and the constant region
heavy chain (SEQ ID NO: 589) and constant region light chain (SEQ
ID NO: 587) sequences. This embodiment further contemplates nucleic
acids encoding variants comprising at least one amino acid
substitutions or deletions to the variable heavy and variable light
chain sequences respectively set forth in SEQ ID NO: 657 and SEQ ID
NO: 709 and the heavy chain and light chain constant regions
respectively set forth in SEQ ID NO: 588 and SEQ ID NO: 586, that
do not substantially affect IL-6 binding and/or desired effector
function.
[0061] In an embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof is aglycosylated
or substantially aglycosylated, e.g., as a result of one or more
modifications in the Fc region of the antibody.
[0062] In an embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof contain an Fc
region that has been modified to alter effector function,
half-life, proteolysis, and/or glycosylation. Preferably the Fc
region is modified to eliminate glycosylation.
[0063] In an embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof is a human,
humanized, single chain or chimeric antibody.
[0064] In an embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof is a humanized
antibody derived from a rabbit (parent) anti-IL-6 antibody.
[0065] In an embodiment of the subject technology, the framework
regions (FRs) in the variable light region and the variable heavy
regions of said anti-IL-6 antibody or antibody fragment or variant
thereof respectively is human FRs which are unmodified or which
have been modified by the substitution of at most 2 or 3 human FR
residues in the variable light or heavy chain region with the
corresponding FR residues of the parent rabbit antibody, and the
human FRs have been derived from human variable heavy and light
chain antibody sequences which have been selected from a library of
human germline antibody sequences based on their high level of
homology to the corresponding rabbit variable heavy or light chain
regions relative to other human germline antibody sequences
contained in the library. As disclosed in detail infra in a
preferred embodiment the antibody will comprise human FRs which are
selected based on their high level of homology (degree of sequence
identity) to that of the parent antibody that is humanized.
[0066] In one embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof includes a heavy
chain polypeptide sequence comprising: SEQ ID NO: 3, 18, 19, 652,
656, 657, 658, 661, 664, 665, 704, or 708; and may further comprise
a V.sub.L polypeptide sequence comprising: SEQ ID NO: 2, 20, 647,
651, 660, 666, 699, 702, 706, or 709 or a variant thereof wherein
at least one of the framework residues (FR residues) in said
V.sub.H or V.sub.L polypeptide may have been substituted with
another amino acid residue resulting in an anti-IL-6 antibody or
antibody fragment or variant thereof that specifically binds human
IL-6, or includes a polypeptide wherein the CDRs therein are
incorporated into a human framework homologous to said sequence.
Preferably the variable heavy and light sequences comprise those in
SEQ ID NO: 657 and 709, respectively.
[0067] In an embodiment of the subject technology, at least one of
said FR residues may be substituted with an amino acid present at
the corresponding site in a parent rabbit anti-IL-6 antibody from
which the complementarity determining regions (CDRs) contained in
said V.sub.H or V.sub.L polypeptides have been derived or by a
conservative amino acid substitution.
[0068] In an embodiment of the subject technology, said anti-IL-6
antibody, or antibody fragment or variant thereof, is
humanized.
[0069] In an embodiment of the subject technology, said anti-IL-6
antibody, or antibody fragment or variant thereof, is chimeric.
[0070] In an embodiment of the subject technology, said anti-IL-6
antibody, or antibody fragment or variant thereof, further includes
a human Fc, e.g., an Fc region comprised of the variable heavy and
light chain constant regions set forth in SEQ ID NO: 704 and
702.
[0071] In an embodiment of the subject technology, said human Fc
may be derived from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8,
IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18
or IgG19.
[0072] In an embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof includes a
polypeptide having at least about 90% sequence homology to at least
one of the polypeptide sequences of SEQ ID NO: 3, 18, 19, 652, 656,
657, 658, 661, 664, 665, 704, 708, 2, 20, 647, 651, 660, 666, 699,
702, 706, or 709.
[0073] In an embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof has an elimination
half-life of at least about 30 days.
[0074] The subject technology also contemplates the administration
of conjugates of anti-IL-6 antibodies and humanized, chimeric or
single chain versions thereof and other binding fragments and
variants thereof conjugated to at least one functional or
detectable moieties.
[0075] In an embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof is directly or
indirectly attached to a detectable label or therapeutic agent.
[0076] In one embodiment, the IL-6 antagonist is an antisense
nucleic acid. In another embodiment of the subject technology, the
IL-6 antagonist is an antisense nucleic acid, for example
comprising at least approximately 10 nucleotides of a sequence
encoding IL-6, IL-6 receptor alpha, gp130, p38 MAP kinase, JAK1,
JAK2, JAK3, or SYK. In a further embodiment of the subject
technology, the antisense nucleic acid includes DNA, RNA, peptide
nucleic acid, locked nucleic acid, morpholino (phosphorodiamidate
morpholino oligo), glycerol nucleic acid, threose nucleic acid, or
any combination thereof.
[0077] In one embodiment, the IL-6 antagonist includes Actemra.RTM.
(Tocilizumab), Remicade.RTM., Zenapax.RTM. (daclizumab), or any
combination thereof.
[0078] In one embodiment, the IL-6 antagonist includes a
polypeptide having a sequence comprising a fragment of IL-6, IL-6
receptor alpha, gp130, p38 MAP kinase, JAK1, JAK2, JAK3, SYK, or
any combination thereof, such as a fragment or full-length
polypeptide that is at least 40 amino acids in length. In another
embodiment of the subject technology, the IL-6 antagonist includes
a soluble IL-6, IL-6 receptor alpha, gp130, p38 MAP kinase, JAK1,
JAK2, JAK3, SYK, or any combination thereof.
[0079] In an embodiment of the subject technology, the IL-6
antagonist is coupled to a half-life increasing moiety.
[0080] In another aspect the subject technology provides novel
pharmaceutical compositions and their use in novel combination
therapies and comprising administration of an anti-IL-6 antibody,
such as any one of Ab1-Ab36 antibodies described in Table 1 or a
fragment or variant thereof, and at least one other therapeutic
compound such as an anti-cytokine agent.
[0081] In an embodiment of the subject technology, the IL-6
antagonist may target IL-6, IL-6 receptor alpha, gp130, p38 MAP
kinase, JAK1, JAK2, JAK3, SYK, or any combination thereof. In one
embodiment, the IL-6 antagonist includes an antibody, an antibody
fragment, a peptide, a glycoalkoid, an antisense nucleic acid, a
ribozyme, a retinoid, an avemir, a small molecule, or any
combination thereof. In one embodiment, the IL-6 antagonist
includes an anti-IL-6R, anti-gp130, anti-p38 MAP kinase, anti-JAK1,
anti-JAK2, anti-JAK3, or anti-SYK antibody or antibody fragment. In
an embodiment of the subject technology, the antagonist includes an
anti-IL-6 antibody (e.g., any one of Ab1-Ab36 antibodies described
in Table 1) or antibody fragment or variant thereof.
[0082] The present subject technology also pertains to methods of
improving survivability or quality of life of a patient having or
at risk of developing psoriatic arthritis comprising administering
to the patient an anti-IL-6 antibody (e.g., ALD518 antibody) or
antibody fragment or variant thereof, whereby the patient's
C-reactive protein ("CRP") level is lowered.
[0083] In one embodiment of the subject technology, the anti-IL-6
antibody or antibody fragment or variant thereof is administered to
the patient with a frequency at most once per period of
approximately 4, 8, 12, 16, 20, or 24 weeks.
[0084] In an embodiment of the subject technology, the patient's
quality of life is improved.
[0085] This subject technology relates to novel anti-IL-6
antibodies, novel therapies and therapeutic protocols utilizing
anti-IL-6 antibodies, and pharmaceutical formulations containing
anti-IL-6 antibodies. In preferred embodiments, an anti-IL-6
antibody is any one of Ab1-Ab36 antibodies described in Table 1,
which includes rabbit or humanized forms thereof, as well as heavy
chains, light chains, fragments, variants, and CDRs thereof, or an
antibody or antibody fragment that specifically binds to the same
linear or conformational epitope(s) on an intact human IL-6
polypeptide fragment thereof as Ab1. The subject application
pertains in particular to preferred formulations and therapeutic
uses of an exemplary humanized antibody referred to herein as any
one of Ab1-Ab36 antibodies described in Table 1 and variants
thereof. In preferred embodiments, the anti-IL-6 antibody has an in
vivo half-life of at least about 30 days, has an in vivo effect of
lowering C-reactive protein, possesses a binding affinity (Kd) for
IL-6 of less than about 50 picomolar, and/or has a rate of
dissociation (K.sub.off) from IL-6 of less than or equal to
10.sup.-4 S.sup.-1.
[0086] In one aspect, this subject technology pertains to methods
of improving survivability or quality of life of a patient in need
thereof, comprising administering to a patient with or at risk of
developing psoriatic arthritis as a result of disease or a
therapeutic regimen comprising the administration of an anti-IL-6
antibody, such as any one of Ab1-Ab36 antibodies described in Table
1 antibody or a fragment or variant thereof (e.g., Ab1).
[0087] In this embodiment, anti-IL-6 antibodies, or antigen-binding
fragments thereof is administered at effective doses to less
inflammation, pain, and loss of mobility experienced from psoriatic
arthritis, e.g., dosages ranging from about 25-500 mg, preferably
at least about 25, 80, 100, 120, 160, 200, 240, or 320 mg dosages.
In an embodiment, the effective dosage ranges between about 25 to
160 mg/4 weeks, per person, delivered to a subject in need thereof
by a subcutaneous injection.
[0088] Another embodiment relates to methods of improving
survivability or quality of life of a patient diagnosed with
psoriatic arthritis, comprising administering to the patient an
anti-IL-6 antibody or antigen-binding fragment or variant thereof,
whereby the patient's serum C-reactive protein ("CRP") level is
stabilized and preferably reduced, and monitoring the patient to
assess the reduction in the patient's serum CRP level. In an
embodiment, the patient has an elevated C-reactive protein (CRP)
level prior to treatment. In an embodiment, the patient may have an
elevated serum CRP level prior to treatment.
[0089] In an embodiment of the subject technology, the patient's
serum CRP level remains decreased for an entire period intervening
two consecutive anti-IL-6 antibody administrations.
[0090] In one embodiment, the patient may have been diagnosed
psoriatic arthritis.
[0091] In one embodiment, the antibody, or antigen-binding fragment
thereof, is engineered, e.g., produced by genetic engineering
methods such as having been expressed from a recombinant cell. In
another embodiment, the cell may be selected from a mammalian,
yeast, bacterial, and insect cell. In another embodiment, the cell
may be a yeast cell. In another embodiment, the cell is a diploidal
yeast cell. In another embodiment, the yeast cell is a Pichia
yeast. In another embodiment, the anti-IL-6 antibody is produced in
a yeast based (Pichia pastoris) expression system using
conventional fermentation processes and downstream purification. In
one embodiment, the antibodies and antibody fragments described
herein is expressed in yeast cells. In one embodiment, the mating
competent yeast is a member of the Saccharomycetaceae family, which
includes the genera Arxiozyma; Ascobotryozyma; Citeromyces;
Debaryomyces; Dekkera; Eremothecium; Issatchenkia; Kazachstania;
Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia;
Saccharomyces; Saturnispora; Tetrapisispora; Torulaspora;
Williopsis; and Zygosaccharomyces. Other types of yeast potentially
useful in the subject technology include Yarrowia, Rhodosporidium,
Candida, Hansenula, Filobasium, Filobasidellla, Sporidiobolus,
Bullera, Leucosporidium, and Filobasidella. In a preferred
embodiment, the mating competent yeast may a member of the genus
Pichia. In a further preferred embodiment, the mating competent
yeast of the genus Pichia is one of the following species: Pichia
pastoris, Pichia methanolica, and Hansenula polymorphs (Pichia
angusta). In a particularly preferred embodiment, the mating
competent yeast of the genus Pichia may the species Pichia
pastoris.
[0092] In exemplary embodiments the invention comprises or consists
of a method for treating or preventing psoriatic or rheumatoid
arthritis, or managing one or more of the symptoms of psoriatic or
rheumatoid arthritis comprising administration of a composition
comprising an effective amount of an anti-IL-6 antibody or antibody
fragment thereof to a subject in need thereof, wherein the
anti-IL-6 antibody or antibody fragment thereof comprises a
variable light (V.sub.L) chain polypeptide comprising a CDR1
sequence of SEQ ID NO:4, a CDR2 sequence of SEQ ID NO:5, and a CDR3
sequence of SEQ ID NO:6, and a variable heavy (V.sub.H) chain
polypeptide comprising a CDR1 sequence of SEQ ID NO:7, a CDR2
sequence of SEQ ID NOs:8 or 120, and a CDR3 sequence of SEQ ID
NO:9.
[0093] In another exemplary embodiments the invention comprises or
consists of a method for treating or preventing psoriatic or
rheumatoid arthritis or managing one or more of the symptoms of
psoriatic arthritis, comprising administration of a composition
comprising an effective amount of an anti-IL-6 antibody or antibody
fragment thereof to a subject in need thereof, wherein the
anti-IL-6 antibody or antibody fragment thereof comprises a
variable light (V.sub.L) chain polypeptide comprising the amino
acid sequence in SEQ ID NO:20, 702 or 709, and a variable heavy
(V.sub.H) chain polypeptide comprising the amino acid sequence in
SEQ ID NO:18, 19, 657 or 704.
[0094] In another exemplary embodiments the invention comprises or
consists of a method for treating or preventing psoriatic or
rheumatoid arthritis or managing one or more of the symptoms of
psoriatic arthritis of claim 1, comprising administration of a
composition comprising an effective amount of an anti-IL-6 antibody
or antibody fragment thereof to a subject in need thereof, wherein
the anti-IL-6 antibody or antibody fragment thereof comprises a
variable light (V.sub.L) chain polypeptide comprising the amino
acid sequence in SEQ ID NO:20 or 709, and a variable heavy
(V.sub.H) chain polypeptide comprising the amino acid sequence in
SEQ ID NO:18, 19, or 657.
[0095] In yet another exemplary embodiments the invention comprises
or consists of a method for treating or preventing psoriatic or
rheumatoid arthritis or managing one or more of the symptoms of
psoriatic arthritis, comprising administration of a composition
comprising an effective amount of an anti-IL-6 antibody or antibody
fragment thereof to a subject in need thereof, wherein the
anti-IL-6 antibody or antibody fragment thereof comprises a light
chain polypeptide comprising the polypeptide having the amino acid
sequence in SEQ ID NO:702 and a heavy chain comprising the
polypeptide having the amino acid sequence of SEQ ID NO:704.
[0096] In any of the foregoing embodiments said antibody fragment
may e.g., be a Fab fragment, a Fab' fragment, a F(ab')2 fragment,
an scFv, a camelbody, a nanobody, a MetMab like monovalent agent,
or an IgNAR (single-chain antibodies derived from sharks).
[0097] In yet another exemplary embodiments the invention comprises
or consists of a method for treating or preventing psoriatic
arthritis or managing one or more of the symptoms of psoriatic
arthritis, comprising administration of a composition comprising an
anti-IL-6 antibody or antibody fragment thereof comprises a V.sub.L
chain polypeptide at least 80% identical to the amino acid sequence
of SEQ ID NO:709, and/or a V.sub.H chain polypeptide at least 80%
identical to the amino acid sequence of SEQ ID NO:657.
[0098] In yet another exemplary embodiments the invention comprises
or consists of a method for treating or preventing psoriatic or
rheumatoid arthritis or managing one or more of the symptoms of
psoriatic or rheumatoid arthritis, comprising administration of a
composition comprising an anti-IL-6 antibody or antibody fragment
thereof which comprises a V.sub.L chain polypeptide at least 95%
identical to the amino acid sequence of SEQ ID NO:709, and/or a
V.sub.H chain polypeptide at least 95% identical to the amino acid
sequence of SEQ ID NO:657.
[0099] In yet another exemplary embodiments the invention comprises
or consists of a method for treating or preventing psoriatic or
rheumatoid arthritis or managing one or more of the symptoms of
psoriatic arthritis, comprising administration of a composition
comprising an anti-IL-6 antibody or antibody fragment thereof which
comprises a V.sub.L chain polypeptide at least 95% identical to the
amino acid sequence of SEQ ID NO:709, and/or a V.sub.H chain
polypeptide at least 95% identical to the amino acid sequence of
SEQ ID NO:657.
[0100] In yet another exemplary embodiments the invention comprises
or consists of a method for treating or preventing psoriatic or
rheumatoid arthritis or managing one or more of the symptoms of
psoriatic or rheumatoid arthritis, comprising administration of a
composition comprising an anti-IL-6 antibody or antibody fragment
thereof which comprises a V.sub.L chain polypeptide identical to
the amino acid sequence of SEQ ID NO:709, and/or a V.sub.H chain
polypeptide identical to the amino acid sequence of SEQ ID
NO:657.
[0101] In yet another exemplary embodiments the invention comprises
or consists of a method for treating or preventing psoriatic
arthritis or managing one or more of the symptoms of psoriatic
arthritis, comprising administration of a composition comprising an
anti-IL-6 antibody or antibody fragment thereof which comprises a
light chain polypeptide at least 90, 95 or 99% identical to the
amino acid sequence of SEQ ID NO:702, and/or a heavy chain
polypeptide at least 90, 95 or 99% identical to the amino acid
sequence of SEQ ID NO:704.
[0102] In any of the foregoing exemplary embodiments, said
anti-IL-6 antibody or antibody fragment thereof is
aglycosylated.
[0103] In any of the foregoing exemplary embodiments said antibody
or antibody fragment comprises an Fc region that has been modified
to alter effector function, half-life, proteolysis, and/or
glycosylation.
[0104] In any of the foregoing exemplary embodiments wherein said
antibody or antibody fragment comprises a human Fc derived from
IgG1, IgG2, IgG3, or IgG4.
[0105] In any of the foregoing exemplary embodiments said antibody
or antibody fragment thereof is a human, humanized, single chain or
chimeric antibody.
[0106] In any of the foregoing exemplary embodiments said antibody
or antibody fragment specifically binds to human cell surface
expressed IL-6 and/or to circulating soluble IL-6 molecules in
vivo.
[0107] In any of the foregoing exemplary embodiments said antibody
or antibody fragment specifically binds to IL-6 expressed on or by
human cells in the subject.
[0108] In any of the foregoing exemplary embodiments said antibody
or antibody fragment has an in vivo half-life of at least about 30
days in a healthy human subject.
[0109] In any of the foregoing exemplary embodiments said antibody
or antibody fragment has a binding affinity (Kd) for IL-6 of less
than about 50 picomolar, or a rate of dissociation (K.sub.off) from
IL-6 of less than or equal to 10.sup.-4 S.sup.-1.
[0110] In any of the foregoing exemplary embodiments said antibody
or antibody fragment specifically binds to the same linear or
conformational epitope(s) and/or competes for binding to the same
linear or conformational epitope(s) on an intact human IL-6
polypeptide or fragment thereof as an anti-IL-6 antibody comprising
the polypeptides of SEQ ID NO: 702 and SEQ ID NO: 704.
[0111] In any of the foregoing exemplary embodiments said antibody
or antibody fragment contains an Fc region that has been modified
to alter effector function, half-life, proteolysis, and/or
glycosylation.
[0112] In any of the foregoing exemplary embodiments a single
dosage effective amount of aid anti-IL-6 antibody or antibody
fragment comprises at least or consists of 1, 5, 10, 15, 20, 25,
50, 60, 80, 100, 120, 160, 200, or 320 mg of said anti-IL-6
antibody or antibody fragment.
[0113] In any of the foregoing exemplary embodiments a dosage
effective amount of said anti-IL-6 antibody or antibody fragment is
between about 0.1 and 100 mg/kg of body weight of the subject.
[0114] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment thereof is administered at least 1, 2, 3, 4,
or 5 doses.
[0115] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen that comprises or consists of administering said anti-IL-6
antibody or antibody fragment every 4 weeks or every month.
[0116] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen that comprises or consists of administering 1, 5, 10, 15,
20 or 25 mg of said anti-IL-6 antibody or antibody fragment every 4
weeks or every month.
[0117] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen that comprises or consists of administering 25 mg of said
anti-IL-6 antibody or antibody fragment every 4 weeks or every
month.
[0118] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen that comprises or consists of administering 25 mg of said
anti-IL-6 antibody or antibody fragment every 4 weeks.
[0119] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen is effected every 4 weeks or monthly for 8, 12, 16, 20, 24,
28, 32, 36 weeks or more or for 2, 3, 4, 5, 6 or more months.
[0120] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen wherein said subject is treated by a dosage regimen that
comprises or consists of administering 50, 60 or 75 mg of said
anti-IL-6 antibody or antibody fragment every 4 weeks or every
month.
[0121] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen The method of any of the foregoing claims, wherein said
subject is treated by a dosage regimen that comprises or consists
of administering 80, 100 or 120 mg of said anti-IL-6 antibody or
antibody fragment every 4 weeks or every month.
[0122] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen wherein said subject is treated by a dosage regimen that
comprises or consists of administering 160 mg of said anti-IL-6
antibody or antibody fragment every 4 weeks or every month.
[0123] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen that comprises or consists of administering 200 mg of said
anti-IL-6 antibody or antibody fragment every 4 weeks or every
month.
[0124] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen wherein said subject is subject is treated by a dosage
regimen that comprises or consists of administering said anti-IL-6
antibody or antibody fragment every 4 weeks or every month for at
least 16 weeks or 4 months.
[0125] In any of the foregoing exemplary embodiments said anti-IL-6
antibody or fragment composition is administered in a dosage
regimen wherein said subject is subject is treated by a dosage
regimen that comprises or consists of administering said anti-IL-6
antibody or antibody fragment every 4 weeks or every month for at
least 20 or 24 weeks or at least 5 or 6 months.
[0126] In any of the foregoing exemplary embodiments the treated
patient or subject has elevated C-reactive protein ("CRP").
[0127] In any of the foregoing exemplary embodiments the treated
patient or subject has elevated IL-6 serum level.
[0128] In any of the foregoing exemplary embodiments the treated
patient or subject has elevated IL-6 level in the joints.
[0129] In any of the foregoing exemplary embodiments the treated
patient or subject has had an inadequate response to non-steroidal
anti-inflammatory drugs (NSAIDs).
[0130] In any of the foregoing exemplary embodiments the treated
patient or subject has had an inadequate response to non-biologic
Disease Modifying Anti-Rheumatic Drugs (DMARDs).
[0131] In any of the foregoing exemplary embodiments said antibody
or antibody fragment thereof inhibits with at least one activity
associated with IL-6.
[0132] In any of the foregoing exemplary embodiments the antibody
or antibody fragment inhibits at least one of the at least one
activity associated with IL-6 is an in vitro activity comprising
stimulation of proliferation of T1165 cells; binding of IL-6 to
IL-6R; activation (dimerization) of the gp130 signal-transducing
glycoprotein; formation of IL-6/IL-6R/gp130 multimers; stimulation
of haptoglobin production by HepG2 cells modified to express human
IL-6 receptor; or any combination thereof.
[0133] In any of the foregoing exemplary embodiments the treated
patient or subject prior to administration of the antibody, or
antigen-binding fragment thereof, the subject has exhibited or is
at risk for developing at least one of the following symptoms:
elevated serum C-reactive protein ("CRP"); elevated erythrocyte
sedimentation rate; or a combination thereof.
[0134] In any of the foregoing exemplary embodiments the antibody
or antibody fragment thereof is directly or indirectly coupled to a
detectable label, cytotoxic agent, therapeutic agent, or an
immunosuppressive agent.
[0135] In any of the foregoing exemplary embodiments the antibody
or antibody fragment thereof is directly or indirectly coupled to a
detectable label comprising fluorescent dyes, bioluminescent
materials, radioactive materials, chemiluminescent moieties,
streptavidin, avidin, biotin, radioactive materials, enzymes,
substrates, horseradish peroxidase, acetylcholinesterase, alkaline
phosphatase, .quadrature.-galactosidase, luciferase, rhodamine,
fluorescein, fluorescein isothiocyanate, umbelliferone,
dichlorotriazinylamine, phycoerythrin, dansyl chloride, luminol,
luciferin, aequorin, Iodine 125 (1251), Carbon 14 (14C), Sulfur 35
(35S), Tritium (3H), Phosphorus 32 (32P), or any combination
thereof.
[0136] In any of the foregoing exemplary embodiments the antibody
or antibody fragment thereof is co-administered with another
therapeutic agent selected from the group consisting of analgesics,
antibiotics, anti-cachexia agents, anti-coagulants, anti-cytokine
agents, antiemetic agents, anti-fatigue agent, anti-fever agent,
anti-inflammatory agents, anti-nausea agents, antipyretics,
antiviral agents, anti-weakness agent, chemotherapy agents,
cytokine antagonist, cytokines, cytotoxic agents, gene therapy
agents, growth factor, IL-6 antagonists, immunosuppressive agents,
statins, or any combination thereof.
[0137] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is co-administered with an agonist of a factor
comprising tumor necrosis factor-alpha, interferon gamma,
interleukin 1 alpha, interleukin 1 beta, interleukin 6, or any
combination thereof.
[0138] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is co-administered with a cytokine antagonist
which is an antagonist of TNF-.alpha., IL-1.beta., IL-1.alpha.,
IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-.alpha.,
IFN-.beta., IFN-.gamma., BAFF, CXCL13, IP-10, leukemia-inhibitory
factor, or a combination thereof.
[0139] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is co-administered with an antagonist of VEGF,
EPO, EGF, HRG, Hepatocyte Growth Factor (HGF), Hepcidin, or any
combination thereof.
[0140] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is co-administered with other anti-IL-6
antibodies or antigen-binding fragments thereof, antisense nucleic
acids, polypeptides, small molecules, or any combination
thereof.
[0141] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is co-administered with an antisense nucleic
acid comprises at least approximately 10 nucleotides of a sequence
encoding IL-6, IL-6 receptor alpha, gp130, p38 MAP kinase, JAK1,
JAK2, JAK3, or SYK.
[0142] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is co-administered with an IL-6 antagonist
polypeptide that comprises a fragment of a polypeptide having a
sequence selected from the group consisting IL-6, IL-6 receptor
alpha, gp130, p38 MAP kinase, JAK1, JAK2, JAK3, and SYK.
[0143] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is co-administered with an IL-6 antagonist
comprising a soluble IL-6, IL-6 receptor alpha, gp130, p38 MAP
kinase, JAK1, JAK2, JAK3, SYK, or any combination thereof, e.g.,
one coupled to a half-life increasing moiety.
[0144] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is administered to the subject in the form of
at least one nucleic acid that encodes said antibody or antibody
fragment thereof.
[0145] In any of the foregoing exemplary embodiments the antibody
or antibody fragment is in a pharmaceutical composition comprising
a pharmaceutical excipient.
[0146] In another exemplary embodiment the invention is directed to
a dosage composition, or syringe or injector pen containing a
single dosage of an anti-IL-6 antibody or antibody fragment which
is for use in treating or preventing psoriatic arthritis according
to any of the foregoing claims, and wherein said anti-IL-6 antibody
or antibody fragment comprises or consists of CDRs, variable heavy
or light polypeptides or light and heavy polypeptides having the
amino acid sequences as set forth in any of the foregoing claims,
wherein the single dosage of said anti-IL-6 antibody or antibody
fragment contained in said composition or syringe or injector pen
containing same comprises at most or consists of 1, 5, 10, 15, 20,
or 25 mg of said anti-IL-6 antibody or antibody fragment.
[0147] In another exemplary embodiment the invention is directed to
a single dosage composition, or syringe or injector pen comprising
an anti-IL-6 antibody or fragment, which is for administration
every 4 weeks or monthly for treating or managing the symptoms of
psoriatic arthritis.
[0148] In another exemplary embodiment the invention is directed to
a single dosage composition, syringe or injector pen of claim which
contains an antibody dosage comprising or consisting of 25 mg of
said anti- an anti-IL-6 antibody or antibody fragment according to
the invention.
[0149] In another exemplary embodiment the invention is directed to
a single dosage composition, syringe or injector pen of claim which
contains an antibody dosage comprising or consisting of 25 mg of
said anti- an anti-IL-6 antibody or antibody fragment according to
the invention, wherein the antibody or antibody fragment is
comprised in an aqueous or 0.9% saline solution.
[0150] In another exemplary embodiment the invention is directed to
a therapeutic regimen for treating or preventing psoriatic
arthritis or managing the side effects of psoriatic arthritis in a
subject in need thereof, wherein the therapeutic regimen comprises
or consists of administering a single dosage of an anti-IL-6
antibody or antibody fragment every 4 weeks or monthly using a
syringe or injector pen which single dosage comprises at most or
consists of 1, 5, 10, 15, 20, or 25 mg of--an anti-IL-6 antibody or
antibody fragment comprising or consisting of any of the anti-IL-6
antibody sequences set forth herein, preferably an anti-IL-6
antibody or antibody fragment that comprises the VL polypeptide of
SEQ ID NO:20 or 709 and a V.sub.H polypeptides having the amino
acid sequence of SEQ ID NO:18, 19 or 657, or that comprises the VL
polypeptide of SEQ ID NO:709 and a V.sub.H polypeptides having the
amino acid sequence of SEQ ID NO:657 or which comprises a light
chain polypeptide having the amino acid sequence of SEQ ID NO:702
and a heavy chain polypeptide having the amino acid sequence of SEQ
ID NO:704.
[0151] In another exemplary embodiment the invention is directed
methods or regimens as above-described wherein the treated subject
or caregiver subcutaneously administers the single dosage every 4
weeks or monthly.
[0152] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
or caregiver subcutaneously administers a single dosage of
anti-IL-6 antibody according to the invention every 4 weeks or
monthly by use of an injector pen.
[0153] In another exemplary embodiment the invention is directed to
methods or regimens as above-described which further include the
administration of a DMARD, a corticosteroid.
[0154] In another exemplary embodiment the invention is directed to
methods or regimens as above-described which further include the
administration of methotrexate.
[0155] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
has developed a resistance or tolerance to methotrexate.
[0156] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
has previously received another anti-IL-6 antagonist or an anti-TNF
biologic.
[0157] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
has previously received Humira.RTM., Remicade.RTM., or
Actmera.RTM..
[0158] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
is treated for at least 12 weeks or 3 months.
[0159] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
is treated for at least 16 weeks or 4 months.
[0160] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
is treated for at least 20 weeks or 5 months.
[0161] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
is treated for at least 24 weeks or 6 months.
[0162] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
is treated for more than at least 24 weeks or 6 months.
[0163] In another exemplary embodiment the invention is directed to
methods or regimens as above-described wherein the treated subject
exhibits prolonged disease remission after said antibody
treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0164] FIG. 1 shows alignments of variable light and variable heavy
sequences between a rabbit antibody variable light and variable
heavy sequences and homologous human sequences and the humanized
sequences. Framework regions are identified FR1-FR4.
Complementarity determining regions are identified as CDR1-CDR3.
Amino acid residues are numbered as shown. The initial rabbit
sequences are called RbtV.sub.L and RbtV.sub.H for the variable
light and variable heavy sequences respectively. Three of the most
similar human germline antibody sequences, spanning from Framework
1 through to the end of Framework 3, are aligned below the rabbit
sequences. The human sequence that is considered the most similar
to the rabbit sequence is shown first. In this example those most
similar sequences are L12A for the light chain and 3-64-04 for the
heavy chain. Human CDR3 sequences are not shown. The closest human
Framework 4 sequence is aligned below the rabbit Framework 4
sequence. The vertical dashes indicate a residue where the rabbit
residue is identical with at least one of the human residues at the
same position. The bold residues indicate that the human residue at
that position is identical to the rabbit residue at the same
position. The final humanized sequences are called V.sub.Lh and
V.sub.Hh for the variable light and variable heavy sequences
respectively. The underlined residues indicate that the residue is
the same as the rabbit residue at that position but different than
the human residues at that position in the three aligned human
sequences.
[0165] FIGS. 2 and 3 show alignments between a rabbit antibody
light and variable heavy sequences and homologous human sequences
and the humanized sequences. Framework regions are identified as
FR1-FR4. Complementarity determining regions are identified as
CDR1-CDR3.
[0166] FIGS. 4A-B and 5A-B show alignments between light and
variable heavy sequences, respectively, of different forms of Ab1.
Framework regions are identified as FR1-FR4. Complementarity
determining regions are identified as CDR1-CDR3. Sequence
differences within the CDR regions highlighted.
[0167] FIG. 6 provides a pharmacokinetic profile of antibody Ab1 in
cynomolgus monkey. Plasma levels of antibody Ab1 were quantitated
through antigen capture ELISA. This protein displays a half-life of
between 12 and 17 days consistent with other full length humanized
antibodies.
[0168] FIG. 7A-D provides binding data for antibodies Ab4, Ab3, Ab8
and Ab2, respectively. FIG. 7E provides binding data for antibodies
Ab1, Ab6 and Ab7.
[0169] FIG. 8 shows the mean plasma concentration of Ab1 resulting
from a single administration of Ab1 to patients with advanced
cancer.
[0170] FIG. 9A demonstrates suppression of serum CRP levels in
healthy individuals.
[0171] FIG. 9B demonstrates suppression of serum CRP levels in
advanced cancer patients.
[0172] FIG. 10 shows the mean CRP values for each dosage
concentrations (placebo, 80 mg, 160 mg, and 320 mg) of the Ab1
monoclonal antibody.
[0173] FIG. 11 shows the change in median values of CRP from each
dosage concentration group corresponding to FIG. 10.
[0174] FIG. 12 shows a reduction in serum CRP levels in patients
with various cancers after dosing at 80, 160 or 320 mg for 12
weeks.
[0175] FIG. 13 shows the effect of subcutaneous and intravenous
administration of ALD518 through week 12 after antibody dosing at
50 or 100 mg.
[0176] FIG. 14 shows the effect of subcutaneous and intravenous
administration of ALD518 through week 12 after antibody dosing at
50 or 100 mg.
[0177] FIG. 15 shows plasma CRP level concentrations after
subcutaneous or intravenous dosing of humanized Ab1.
[0178] FIG. 16 shows the study design used in Example 21 for safety
and efficacy studies of Ab1, also known as MBS-945429 or
clazakizumab. Single asterisk (*) indicates that rescue was allowed
during Period II during the Long Term (Open-Label) Extension until
all ongoing subjects were switched to the final dose of 2 mg.
Double asterisks (**) indicate that during Long Term (Open Label)
Extension, subjects continued the doses from Period II until a
final dose was selected based on the Week 24 final analysis. The
selected dose was 25 mg after the Week 24 analysis and was used for
subjects remaining in the study for the rest of the long term
extension.
[0179] FIG. 17 shows a bar plot of ACR20 response by treatment by
clazakizumab at day 113 (Week 16) for all randomized and treated
subjects.
[0180] FIG. 18 shows the ACR20 response rate to clazakizumab at the
scheduled time point during the double-blind period (periods I and
II) in all randomized and treated subjects. As shown, beginning as
early as Day 29 (Week 4) and increasing through Day 169 (Week 24),
subjects in the 3 clazakizumab groups achieved a numerically higher
ACR20 response rate compared with the placebo group. At Day 113
(Week 16) the differences from placebo in ACR 20 response rates
were numerically higher in the 25 mg and 100 mg clazakizumab groups
compared with the difference from placebo and the difference from
placebo with the 200 mg clazakizumab group (as noted previously).
ACR20 response rates continued to improve over time and the
difference from placebo in the 25 mg and 100 mg clazakizumab groups
was again numerically higher compared with the difference from
placebo in the 200 mg clazakizumab group at Day 141 (Week 20) and
Day 169 (Week 24).
[0181] FIG. 19 shows ACR50 response rate to clazakizumab at the
scheduled time point during the double blind (periods I and II) for
all randomized and treated subjections. As shown, beginning as
early as Day 29 (Week 4) and continuing through Day 169 (Week 24),
subjects in all 3 clazakizumab dose groups achieved a numerically
higher ACR50 response rate compared with the placebo group. At Day
113 (Week 16) the difference from placebo was numerically higher in
the 25 mg and 100 mg clazakizumab groups compared with the 200 mg
clazakizumab group. A similar trend was noted at Day 169 (Week 24)
with numerically higher differences from placebo noted in the 25 mg
and 100 mg clazakizumab groups compared to the 200 mg clazakizumab
group.
[0182] FIG. 20 shows ACR70 response rate to clazakizumab at the
scheduled time point during the double blind (periods I and II) for
all randomized and treated subjections. As shown, beginning as
early as Day 57 (Week 8) and continuing through Day 169 (Week 24),
subjects in all 3 clazakizumab groups achieved a higher ACR70
response rate compared with the placebo group. At Day 113 (Week 16)
the difference from placebo was numerically higher in the 25 mg and
100 mg clazakizumab groups compared with the difference from
placebo for the 200 mg clazakizumab group. A similar trend was
noted at Day 169 (Week 24) with numerically higher differences from
placebo noted in the 25 mg and 100 mg clazakizumab groups compared
to the 200 mg clazakizumab group.
[0183] FIG. 21 shows the median percent improvement in tender joint
count after clazakizumab administration and during the double-blind
period (periods I and II) for all randomized and treated subjects).
As shown, beginning at Day 8 and continuing through Day 169 (Week
24), a numerically greater improvement in the median change from
baseline tender joint count was shown for at least 1 dose of
clazakizumab compared with the placebo group. Mean change from
baseline results also showed numerically greater improvement in the
tender joint count for all 3 clazakizumab groups compared with the
placebo group at Day 113 (Week 16) and Day 169 (Week 24).
[0184] FIG. 22 shows the median percent improvement in swollen
joint count after clazakizumab administration and during the
double-blind period (periods I and II) for all randomized and
treated subjects. As shown, beginning at Day 8 and continuing
through Day 169 (Week 24), the median change from baseline swollen
joint count showed numerically greater improvement in all 3
clazakizumab groups compared with the placebo group. Mean change
from baseline results for swollen joint counts also showed
numerically greater improvement in the 3 clazakizumab groups
compared with the placebo group at Day 113 (Week 16) and Day 169
(Week 24).
[0185] FIG. 23 shows the response mean values of total IL-6
biomarker over time by treatment with clazakizumab during the
double-blind period (periods I and II) in all pharmacodynamic
analysis subjects.
[0186] FIG. 24 shows the mean values of free IL-6 biomarker over
time by treatment with clazakizumab during the double-blind period
(periods I and II) in all pharmacodynamic analysis subjects.
[0187] FIG. 25 shows that clazakizumab when used at doses of 25 mg,
100 mg, and 200 mg/month with background MTX, 100 mg and 200 mg
monotherapy) demonstrated efficacy over placebo. In addition, each
of the clazakizumab+MTX doses was associated with more patients
achieving stringent measures of response than with adalimumab+MTX.
Overall, there was not a strong dose response relationship at the
doses tested on ACR20, ACR50, ACR70, DAS28-CRP<2.6, CDAI<2.8,
or SDAI<3.3. Within the range of Cmins achieved with the 25 mg
and higher, the relationship between Cmins and the probability of
achieving an ACR response was relatively flat.
[0188] FIG. 26 examines clazakizumab IL-6/IL-6 soluble receptor
complex inhibition data by ACR20 response. The data indicates that
ACR20 responders had higher levels of inhibition at Week 12 than
non-responders with the 25 mg dose in combination with MTX.
DETAILED DESCRIPTION
Definitions
[0189] It is to be understood that this subject technology is not
limited to the particular methodology, protocols, cell lines,
animal species or genera, and reagents described, as such may vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to limit the scope of the present subject technology which
will be limited only by the appended claims.
[0190] As used herein the singular forms "a", "and", and "the"
include plural referents unless the context clearly dictates
otherwise. Thus, for example, reference to "a cell" includes a
plurality of such cells and reference to "the protein" includes
reference to at least one proteins and equivalents thereof known to
those skilled in the art, and so forth. All technical and
scientific terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which this
subject technology belongs unless clearly indicated otherwise.
[0191] Amplification as used herein refers broadly to the
amplification of polynucleotide sequences is the in vitro
production of multiple copies of a particular nucleic acid
sequence. The amplified sequence is usually in the form of DNA. A
variety of techniques for carrying out such amplification are known
in the art. See, e.g., Van Brunt (1990) Bio/Technol. 8(4): 291-294.
Polymerase chain reaction or PCR is a prototype of nucleic acid
amplification, and use of PCR herein should be considered exemplary
of other suitable amplification techniques.
[0192] Engineered, as used herein with an antibody, refers to a
non-naturally occurring antibody produced by recombinant or genetic
engineering methodologies known in the art or described herein.
[0193] Antibody, as used herein, refers broadly to any polypeptide
chain-containing molecular structure with a specific shape that
fits to and recognizes an epitope, where at least one non-covalent
binding interactions stabilize the complex between the molecular
structure and the epitope. The archetypal antibody molecule is the
immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA,
IgE, IgD, from all sources, e.g., human, rodent, rabbit, cow,
sheep, pig, dog, chicken, are considered to be "antibodies."
Antibodies include but are not limited to chimeric antibodies,
human antibodies and other non-human mammalian antibodies,
humanized antibodies, single chain antibodies (scFvs), camelbodies,
nanobodies, IgNAR (single-chain antibodies derived from sharks),
small-modular immunopharmaceuticals (SMIPs), and antibody fragments
(e.g., Fabs, Fab', F(ab').sub.2.) Numerous antibody coding
sequences have been described; and others may be raised by methods
well-known in the art. See Streltsov, et al. (2005) Protein Sci.
14(11): 2901-9; Greenberg, et al. (1995) Nature 374(6518): 168-173;
Nuttall, et al. (2001) Mol Immunol. 38(4): 313-26;
Hamers-Casterman, et al. (1993) Nature 363(6428): 446-8; Gill, et
al. (2006) Curr Opin Biotechnol. 17(6): 653-8.
[0194] Antigen-binding fragment, as used herein, refers broadly to
a fragment of an antibody which recognizes an antigen (e.g.,
paratopes.) The antigen-binding fragment may comprise a paratope
that may be a small region (e.g., 15-22 amino acids) of the
antibody's Fv region and may contain parts of the antibody's heavy
and light chains. See Goldsby, et al. Antigens (Chapter 3)
Immunology p Ed.) New York: W.H. Freeman and Company, pages
57-75.
[0195] C-Reactive Protein (CRP), as used herein, refers broadly to
a 224 amino acid protein found in the blood that rise in response
to inflammation (e.g., GenBank Protein Accession No. NP_000558 and
SEQ ID NO: 726). CRP also encompasses any pre-pro, pro- and mature
forms of this CRP amino acid sequence, as well as mutants and
variants including allelic variants of this sequence. CRP levels,
e.g. in the serum, liver, or elsewhere in the body, can be readily
measured using routine methods and commercially available reagents,
e.g. ELISA, antibody test strip, immunoturbidimetry, rapid
immunodiffusion, visual agglutination, Western blot, Northern blot
As mentioned above CRP levels may in addition be measured in
patients having or at risk of developing thrombosis according to
the subject technology.
[0196] Coding sequence, as used herein refers broadly to an
in-frame sequence of codons that (in view of the genetic code)
correspond to or encode a protein or peptide sequence. Two coding
sequences correspond to each other if the sequences or their
complementary sequences encode the same amino acid sequences. A
coding sequence in association with appropriate regulatory
sequences may be transcribed and translated into a polypeptide. A
polyadenylation signal and transcription termination sequence will
usually be located 3' to the coding sequence. A "promoter sequence"
is a DNA regulatory region capable of binding RNA polymerase in a
cell and initiating transcription of a downstream (3' direction)
coding sequence. Promoter sequences typically contain additional
sites for binding of regulatory molecules (e.g., transcription
factors) which affect the transcription of the coding sequence. A
coding sequence is "under the control" of the promoter sequence or
"operatively linked" to the promoter when RNA polymerase binds the
promoter sequence in a cell and transcribes the coding sequence
into mRNA, which is then in turn translated into the protein
encoded by the coding sequence. A polynucleotide sequence
"corresponds" to a polypeptide sequence if translation of the
polynucleotide sequence in accordance with the genetic code yields
the polypeptide sequence (i.e., the polynucleotide sequence
"encodes" the polypeptide sequence), one polynucleotide sequence
"corresponds" to another polynucleotide sequence if the two
sequences encode the same polypeptide sequence.
[0197] Complementarity determining region, hypervariable region, or
CDR, as used herein refer broadly to at least one of the
hyper-variable or complementarity determining regions (CDRs) found
in the variable regions of light or heavy chains of an antibody
(See Kabat, E. A. et al. (1987) Sequences of Proteins of
Immunological Interest, National Institutes of Health, Bethesda,
Md.). These expressions include the hypervariable regions as
defined by Kabat et al. ("Sequences of Proteins of Immunological
Interest," Kabat E., et al. (1983) US Dept. of Health and Human
Services) or the hypervariable loops in 3-dimensional structures of
antibodies. Chothia and Leska (1987) J Mol. Biol. 196: 901-917. The
CDRs in each chain are held in close proximity by framework regions
and, with the CDRs from the other chain, contribute to the
formation of the antigen binding site. Within the CDRs there are
select amino acids that have been described as the selectivity
determining regions (SDRs) which represent the critical contact
residues used by the CDR in the antibody-antigen interaction
(Kashmiri (2005) Methods 36:25-34). CDRs for exemplary anti-IL-6
antibodies are provided herein.
[0198] Disease or condition, as used herein, refers broadly to a
disease or condition that a patient has been diagnosed with or is
suspected of having, particularly a disease or condition associated
with elevated IL-6. A disease or condition encompasses, without
limitation thereto, psoriatic arthritis, as well as idiopathic
conditions characterized by symptoms that include elevated
IL-6.
[0199] Effective amount, as used herein, refers broadly to an
amount of an active ingredient that is effective to relieve or
reduce to some extent at least one of the symptoms of the disease
in need of treatment, or to retard initiation of clinical markers
or symptoms of a disease in need of prevention, when the compound
is administered. Thus, an effective amount refers to an amount of
the active ingredient which exhibit effects such as (i) reversing
the rate of progress of a disease; (ii) inhibiting to some extent
further progress of the disease; and/or, (iii) relieving to some
extent (or, preferably, eliminating) at least one symptoms
associated with the disease. The effective amount may be
empirically determined by experimenting with the compounds
concerned in known in vivo and in vitro model systems for a disease
in need of treatment. The context in which the phrase "effective
amount" is used may indicate a particular desired effect. For
example, "an amount of an anti-IL-6 antibody effective to prevent
or treat a hypercoagulable state" and similar phrases refer to an
amount of anti-IL-6 antibody that, when administered to a subject,
will cause a measurable improvement in the subject's coagulation
profile, or prevent, slow, delay, or arrest, a worsening of the
coagulation profile for which the subject is at risk. Similarly,
"an amount of an anti-IL-6 antibody effective to reduce serum CRP
levels" and similar phrases refer to an amount of anti-IL-6
antibody that, when administered to a subject, will cause a
measurable decrease in serum CRP levels, or prevent, slow, delay,
or arrest, an increase in serum CRP levels for which the subject is
at risk. Similarly, "an amount of an anti-IL-6 antibody effective
to increase serum albumin levels" and similar phrases refer to an
amount of anti-IL-6 antibody that, when administered to a subject,
will cause a measurable increase in serum albumin levels, or
prevent, slow, delay, or arrest, a decrease in serum albumin levels
for which the subject is at risk. Similarly, "an amount of an
anti-IL-6 antibody effective to reduce weakness" and similar
phrases refer to an amount of anti-IL-6 antibody that, when
administered to a subject, will cause a measurable decrease in
weakness as determined by the hand grip strength test. Similarly,
"an amount of an anti-IL-6 antibody effective to increase weight"
and similar phrases refer to an amount of anti-IL-6 antibody that,
when administered to a subject, will cause a measurable increase in
a patient's weight. An effective amount will vary according to the
weight, sex, age and medical history of the individual, as well as
the severity of the patient's condition(s), the type of disease(s),
mode of administration, and the like. An effective amount may be
readily determined using routine experimentation, e.g., by
titration (administration of increasing dosages until an effective
dosage is found) and/or by reference to amounts that were effective
for prior patients. Generally, the anti-IL-6 antibodies of the
present subject technology will be administered in dosages ranging
between about 0.1 mg/kg and about 20 mg/kg of the patient's
body-weight.
[0200] Expression Vector, as used herein, refers broadly to a DNA
vectors contain elements that facilitate manipulation for the
expression of a foreign protein within the target host cell.
Conveniently, manipulation of sequences and production of DNA for
transformation is first performed in a bacterial host, e.g. E.
coli, and usually vectors will include sequences to facilitate such
manipulations, including a bacterial origin of replication and
appropriate bacterial selection marker. Selection markers encode
proteins necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not
survive in the culture medium. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
(b) complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media. Exemplary vectors and
methods for transformation of yeast are described, for example, in
Burke, D., Dawson, D., & Stearns, T. (2000). Methods in yeast
genetics: a Cold Spring Harbor Laboratory course manual. Plainview,
N.Y.: Cold Spring Harbor Laboratory Press.
[0201] Folding, as used herein, refers broadly to the
three-dimensional structure of polypeptides and proteins, where
interactions between amino acid residues act to stabilize the
structure. While non-covalent interactions are important in
determining structure, usually the proteins of interest will have
intra- and/or intermolecular covalent disulfide bonds formed by two
cysteine residues. For naturally occurring proteins and
polypeptides or derivatives and variants thereof, the proper
folding is typically the arrangement that results in optimal
biological activity, and can conveniently be monitored by assays
for activity, e.g. ligand binding, enzymatic activity.
[0202] Framework region or FR, as used herein refers broadly to at
least one of the framework regions within the variable regions of
the light and heavy chains of an antibody. See Kabat, et al. (1987)
Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda, Md. These expressions include those
amino acid sequence regions interposed between the CDRs within the
variable regions of the light and heavy chains of an antibody. As
mentioned in the preferred embodiments, the FRs may comprise human
FRs highly homologous to the parent antibody (e.g., rabbit
antibody).
[0203] gp130 (also called Interleukin-6 receptor subunit beta), as
used herein, refers broadly to a transmembrane protein that forms
one subunit of type I cytokine receptors in the IL-6 receptor
family (e.g., 918 precursor amino acid sequence available as
Swiss-Prot Protein Accession No. P40189 and SEQ ID NO: 728). gp130
also encompasses any pre-pro, pro- and mature forms of this amino
acid sequence, such as the mature form encoded by amino acids 23
through 918 of the sequence shown, as well as mutants and variants
including allelic variants of this sequence.
[0204] Heterologous region or domain of a DNA construct, as used
herein, refers broadly to an identifiable segment of DNA within a
larger DNA molecule that is not found in association with the
larger molecule in nature. Thus, when the heterologous region
encodes a mammalian gene, the gene will usually be flanked by DNA
that does not flank the mammalian genomic DNA in the genome of the
source organism. Another example of a heterologous region is a
construct where the coding sequence itself is not found in nature
(e.g., a cDNA where the genomic coding sequence contains introns,
or synthetic sequences having codons different than the native
gene). Allelic variations or naturally-occurring mutational events
do not give rise to a heterologous region of DNA as defined
herein.
[0205] Homology, as used herein, refers broadly to a degree of
similarity between a nucleic acid sequence and a reference nucleic
acid sequence or between a polypeptide sequence and a reference
polypeptide sequence. Homology may be partial or complete. Complete
homology indicates that the nucleic acid or amino acid sequences
are identical. A partially homologous nucleic acid or amino acid
sequence is one that is not identical to the reference nucleic acid
or amino acid sequence. The degree of homology can be determined by
sequence comparison. The term "sequence identity" may be used
interchangeably with "homology."
[0206] Host cell, as used herein, refers broadly to a cell that
contains an expression vector and supports the replication or
expression of the expression vector. Host cells may be prokaryotic
cells such as E. coli, or eukaryotic cells such as yeast, insect
(e.g., SF9), amphibian, or mammalian cells such as CHO, HeLa,
HEK-293 (e.g., cultured cells, explants, and cells in vivo.)
[0207] Isolated, as used herein, refers broadly to material removed
from its original environment in which it naturally occurs, and
thus is altered by the hand of man from its natural environment.
Isolated material may be, for example, exogenous nucleic acid
included in a vector system, exogenous nucleic acid contained
within a host cell, or any material which has been removed from its
original environment and thus altered by the hand of man (e.g.,
"isolated antibody").
[0208] Improved, as used herein, refers broadly to any beneficial
change resulting from a treatment. A beneficial change is any way
in which a patient's condition is better than it would have been in
the absence of the treatment. "Improved" includes prevention of an
undesired condition, slowing the rate at which a condition worsens,
delaying the development of an undesired condition, and restoration
to an essentially normal condition. For example, improvement in
psoriatic arthritis encompasses any decrease in pain, swelling,
joint stiffness, or inflammation, and/or an increase in joint
mobility.
[0209] IL-6 antagonist, as used herein, refers broadly to any
composition that prevents, inhibits, or lessens the effect(s) of
IL-6 signaling. Generally, such antagonists may reduce the levels
or activity of IL-6, IL-6 receptor alpha, gp130, or a molecule
involved in IL-6 signal transduction, or may reduce the levels or
activity complexes between the foregoing (e.g., reducing the
activity of an IL-6/IL-6 receptor complex). Antagonists include
antisense nucleic acids, including DNA, RNA, or a nucleic acid
analogue such as a peptide nucleic acid, locked nucleic acid,
morpholino (phosphorodiamidate morpholino oligo), glycerol nucleic
acid, or threose nucleic acid. See Heasman (2002) Dev Biol. 243(2):
209-14; Hannon and Rossi (2004) Nature 431(7006):371-8; Paul, et
al. (2002) Nat Biotechnol. 20(5):505-8; Zhang, et al. (2005) J Am
Chem Soc. 127(12):4174-5; Wahlestedt, et al. (2000) Proc Natl Acad
Sci USA. 97(10):5633-8; Hanvey, et al. (1992) Science 258
(5087):1481-5; Braasch, et al. (2002) Biochemistry 41(14): 4503-10;
Schoning, et al. (2000) Science 290(5495): 1347-51. In addition
IL-6 antagonists specifically include peptides that block IL-6
signaling such as those described in any of U.S. Pat. Nos.
5,210,075; 6,172,042; 6,599,875; 6,841,533; and 6,838,433. Also,
IL-6 antagonists according to the subject technology may include
p38 MAP kinase inhibitors such as those reported in U.S. Patent
Application No. 2007/0010529 given this kinase's role in cytokine
production and more particularly IL-6 production. Further, IL-6
antagonists according to the subject technology include the
glycoalkaloid compounds reported in U.S. Patent Application
Publication No. 2005/0090453 as well as other IL-6 antagonist
compounds isolatable using the IL-6 antagonist screening assays
reported therein. Other IL-6 antagonists include antibodies, such
as anti-IL-6 antibodies, anti-IL-6 receptor alpha antibodies,
anti-gp130 antibodies, and anti-p38 MAP kinase antibodies including
(but not limited to) the anti-IL-6 antibodies disclosed herein,
Actemra.RTM. (Tocilizumab), Remicade.RTM., Zenapax.RTM.
(daclizumab), or any combination thereof. Other IL-6 antagonists
include portions or fragments of molecules involved in IL-6
signaling, such as IL-6, IL-6 receptor alpha, and gp130, which may
be native, mutant, or variant sequence, and may optionally be
coupled to other moieties (such as half-life-increasing moieties,
e.g. an Fc domain). For example, an IL-6 antagonist may be a
soluble IL-6 receptor or fragment, a soluble IL-6 receptor:Fc
fusion protein, a small molecule inhibitor of IL-6, an anti-IL-6
receptor antibody or antibody fragment or variant thereof,
antisense nucleic acid. Other IL-6 antagonists include avemirs,
such as C326 (Silverman, et al. (2005) Nat Biotechnol. 23(12):
1556-61) and small molecules, such as synthetic retinoid AM80
(tamibarotene) (Takeda, et al. (2006) Arterioscler Thromb Vasc
Biol. 26(5): 1177-83). Such IL-6 antagonists may be administered by
any means known in the art, including contacting a subject with
nucleic acids which encode or cause to be expressed any of the
foregoing polypeptides or antisense sequences.
[0210] Interleukin-6 (IL-6), as used herein, refers broadly to
interleukin-6 (IL-6) encompasses not only the following 212 amino
acid sequence available as GenBank Protein Accession No. NP_000591
(e.g., SEQ ID NO: 1), but also any pre-pro, pro- and mature forms
of this IL-6 amino acid sequence, as well as mutants and variants
including allelic variants of this sequence.
[0211] Interleukin-6 receptor (IL-6R) (IL-6 receptor alpha (IL-6RA)
[CD126], as used herein, refers broadly to 468 amino acid protein
that binds IL-6, a potent pleiotropic cytokine that regulates cell
growth and differentiation and also plays an important role in
immune response (e.g., Swiss-Prot Protein Accession No. P08887 and
SEQ ID NO: 727). IL-6R also includes any pre-pro, pro- and mature
forms of this amino acid sequence, as well as mutants and variants
including allelic variants of this sequence.
[0212] Mammal, as used herein, refers broadly to any and all
warm-blooded vertebrate animals of the class Mammalia, including
humans, characterized by a covering of hair on the skin and, in the
female, milk-producing mammary glands for nourishing the young.
Examples of mammals include but are not limited to alpacas,
armadillos, capybaras, cats, camels, chimpanzees, chinchillas,
cattle, dogs, goats, gorillas, hamsters, horses, humans, lemurs,
llamas, mice, non-human primates, pigs, rats, sheep, shrews,
squirrels, and tapirs. Mammals include but are not limited to
bovine, canine, equine, feline, murine, ovine, porcine, primate,
and rodent species. Mammal also includes any and all those listed
on the Mammal Species of the World maintained by the National
Museum of Natural History, Smithsonian Institution in Washington
DC.
[0213] Nucleic acid or nucleic acid sequence, as used herein,
refers broadly to a deoxy-ribonucleotide or ribonucleotide
oligonucleotide in either single- or double-stranded form. The term
encompasses nucleic acids, i.e., oligonucleotides, containing known
analogs of natural nucleotides. The term also encompasses
nucleic-acid-like structures with synthetic backbones. Unless
otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions) and complementary sequences,
as well as the sequence explicitly indicated. The term nucleic acid
is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and
polynucleotide.
[0214] Operatively linked, as used herein, refers broadly to when
two DNA fragments are joined such that the amino acid sequences
encoded by the two DNA fragments remain in-frame.
[0215] Paratope, as used herein, refers broadly to the part of an
antibody which recognizes an antigen (e.g., the antigen-binding
site of an antibody.) Paratopes may be a small region (e.g., 15-22
amino acids) of the antibody's Fv region and may contain parts of
the antibody's heavy and light chains. See Goldsby, et al. Antigens
(Chapter 3) Immunology (5.sup.th Ed.) New York: W.H. Freeman and
Company, pages 57-75.
[0216] Patient, as used herein, refers broadly to any animal who is
in need of treatment either to alleviate a disease state or to
prevent the occurrence or reoccurrence of a disease state. Also,
"Patient" as used herein, refers broadly to any animal who has risk
factors, a history of disease, susceptibility, symptoms, signs, was
previously diagnosed, is at risk for, or is a member of a patient
population for a disease. The patient may be a clinical patient
such as a human or a veterinary patient such as a companion,
domesticated, livestock, exotic, or zoo animal. The term "subject"
may be used interchangeably with the term "patient".
[0217] Polyploid yeast that stably expresses or expresses a desired
secreted heterologous polypeptide for prolonged time, as used
herein, refers broadly to a yeast culture that secretes said
polypeptide for at least several days to a week, more preferably at
least a month, still more preferably at least about 1-6 months, and
even more preferably for more than a year at threshold expression
levels, typically at least about 10-25 mg/liter and preferably
substantially greater.
[0218] Polyploidal yeast culture that secretes desired amounts of
recombinant polypeptide, as used herein, refers broadly to cultures
that stably or for prolonged periods secrete at least about 10-25
mg/liter of heterologous polypeptide, more preferably at least
about 50-500 mg/liter, and most preferably at least about 500-1000
mg/liter or more.
[0219] Prolonged improvement in coagulation profile, as used
herein, refers broadly to a measurable improvement in the subject's
coagulation profile relative to the initial coagulation profile
(i.e. the coagulation profile at a time before treatment begins)
that is detectable within about a week from when treatment begins
(e.g. administration of an IL-6 antagonist such as Ab1) and remains
improved for a prolonged duration, e.g., at least about 14 days, at
least about 21 days, at least about 28 days, at least about 35
days, at least about 40 days, at least about 50 days, at least
about 60 days, at least about 70 days, at least about 11 weeks, or
at least about 12 weeks from when the treatment begins.
[0220] Prolonged reduction in serum CRP, as used herein, refers
broadly to a measurable decrease in serum CRP level relative to the
initial serum CRP level (i.e. the serum CRP level at a time before
treatment begins) that is detectable within about a week from when
a treatment begins (e.g. administration of an anti-IL-6 antibody)
and remains below the initial serum CRP level for an prolonged
duration, e.g. at least about 14 days, at least about 21 days, at
least about 28 days, at least about 35 days, at least about 40
days, at least about 50 days, at least about 60 days, at least
about 70 days, at least about 11 weeks, or at least about 12 weeks
from when the treatment begins.
[0221] Promoter, as used herein, refers broadly to an array of
nucleic acid sequences that direct transcription of a nucleic acid.
As used herein, a promoter includes necessary nucleic acid
sequences near the start site of transcription, such as, in the
case of a polymerase II type promoter, a TATA element. A promoter
also optionally includes distal enhancer or repressor elements,
which can be located as much as several thousand base pairs from
the start site of transcription. A "constitutive" promoter is a
promoter that is active under most environmental and developmental
conditions. An "inducible" promoter is a promoter that is active
under environmental or developmental regulation.
[0222] Prophylactically effective amount, as used herein, refers
broadly to the amount of a compound that, when administered to a
patient for prophylaxis of a disease or prevention of the
reoccurrence of a disease, is sufficient to effect such prophylaxis
for the disease or reoccurrence. The prophylactically effective
amount may be an amount effective to prevent the incidence of signs
and/or symptoms. The "prophylactically effective amount" may vary
depending on the disease and its severity and the age, weight,
medical history, predisposition to conditions, preexisting
conditions, of the patient to be treated.
[0223] Prophylaxis, as used herein, refers broadly to a course of
therapy where signs and/or symptoms are not present in the patient,
are in remission, or were previously present in a patient.
Prophylaxis includes preventing disease occurring subsequent to
treatment of a disease in a patient. Further, prevention includes
treating patients who may potentially develop the disease,
especially patients who are susceptible to the disease (e.g.,
members of a patent population, those with risk factors, or at risk
for developing the disease).
[0224] Recombinant as used herein, refers broadly with reference to
a product, e.g., to a cell, or nucleic acid, protein, or vector,
indicates that the cell, nucleic acid, protein or vector, has been
modified by the introduction of a heterologous nucleic acid or
protein or the alteration of a native nucleic acid or protein, or
that the cell is derived from a cell so modified. Thus, for
example, recombinant cells express genes that are not found within
the native (non-recombinant) form of the cell or express native
genes that are otherwise abnormally expressed, under expressed or
not expressed at all.
[0225] Selectable Marker, as used herein, refers broadly to a
selectable marker is a gene or gene fragment that confers a growth
phenotype (physical growth characteristic) on a cell receiving that
gene as, for example through a transformation event. The selectable
marker allows that cell to survive and grow in a selective growth
medium under conditions in which cells that do not receive that
selectable marker gene cannot grow. Selectable marker genes
generally fall into several types, including positive selectable
marker genes such as a gene that confers on a cell resistance to an
antibiotic or other drug, temperature when two ts mutants are
crossed or a ts mutant is transformed; negative selectable marker
genes such as a biosynthetic gene that confers on a cell the
ability to grow in a medium without a specific nutrient needed by
all cells that do not have that biosynthetic gene, or a mutagenized
biosynthetic gene that confers on a cell inability to grow by cells
that do not have the wild type gene; and the like. Suitable markers
include but are not limited to ZEOMYCIN.RTM. (zeocin), neomycin,
G418, LYS3, MET1, MET3a, ADE1, ADE3, and URA3.
[0226] Specifically (or selectively) binds to an antibody or
"specifically (or selectively) immunoreactive with," or
"specifically interacts or binds," as used herein, refers broadly
to a protein or peptide (or other epitope), refers, in some
embodiments, to a binding reaction that is determinative of the
presence of the protein in a heterogeneous population of proteins
and other biologics. For example, under designated immunoassay
conditions, the specified antibodies bind to a particular protein
at least two times greater than the background (non-specific
signal) and do not substantially bind in a significant amount to
other proteins present in the sample. Typically a specific or
selective reaction will be at least twice background signal or
noise and more typically more than about 10 to 100 times
background.
[0227] Signs of disease, as used herein, refers broadly to any
abnormality indicative of disease, discoverable on examination of
the patient; an objective indication of disease, in contrast to a
symptom, which is a subjective indication of disease.
[0228] Solid support, support, and substrate, as used herein,
refers broadly to any material that provides a solid or semi-solid
structure with which another material can be attached including but
not limited to smooth supports (e.g., metal, glass, plastic,
silicon, and ceramic surfaces) as well as textured and porous
materials.
[0229] Subjects as used herein, refers broadly to anyone suitable
to be treated according to the present subject technology include,
but are not limited to, avian and mammalian subjects, and are
preferably mammalian. Mammals of the present subject technology
include, but are not limited to, canines, felines, bovines,
caprines, equines, ovines, porcines, rodents (e.g., rats and mice),
lagomorphs, primates, humans. Any mammalian subject in need of
being treated according to the present subject technology is
suitable. Human subjects of both genders and at any stage of
development (i.e., neonate, infant, juvenile, adolescent, adult)
can be treated according to the present subject technology. The
present subject technology may also be carried out on animal
subjects, particularly mammalian subjects such as mice, rats, dogs,
cats, cattle, goats, sheep, and horses for veterinary purposes, and
for drug screening and drug development purposes. "Subjects" is
used interchangeably with "patients."
[0230] Mating competent yeast species, as used herein refers
broadly encompass any diploid or tetraploid yeast which can be
grown in culture. Such species of yeast may exist in a haploid,
diploid, or tetraploid form. The cells of a given ploidy may, under
appropriate conditions, proliferate for indefinite number of
generations in that form. Diploid cells can also sporulate to form
haploid cells. Sequential mating can result in tetraploid strains
through further mating or fusion of diploid strains. In the present
subject technology the diploid or polyploidal yeast cells are
preferably produced by mating or spheroplast fusion.
[0231] Haploid Yeast Cell, as used herein, refers broadly to a cell
having a single copy of each gene of its normal genomic
(chromosomal) complement.
[0232] Polyploid Yeast Cell, as used herein, refers broadly to a
cell having more than one copy of its normal genomic (chromosomal)
complement.
[0233] Diploid Yeast Cell, as used herein, refers broadly to a cell
having two copies (alleles) of essentially every gene of its normal
genomic complement, typically formed by the process of fusion
(mating) of two haploid cells.
[0234] Tetraploid Yeast Cell, as used herein, refers broadly to a
cell having four copies (alleles) of essentially every gene of its
normal genomic complement, typically formed by the process of
fusion (mating) of two haploid cells. Tetraploids may carry two,
three, four, or more different expression cassettes. Such
tetraploids might be obtained in S. cerevisiae by selective mating
homozygotic heterothallic a/a and alpha/alpha diploids and in
Pichia by sequential mating of haploids to obtain auxotrophic
diploids. For example, a [met his] haploid can be mated with [ade
his] haploid to obtain diploid [his]; and a [met arg] haploid can
be mated with [ade arg] haploid to obtain diploid [arg]; then the
diploid [his] x diploid [arg] to obtain a tetraploid prototroph. It
will be understood by those of skill in the art that reference to
the benefits and uses of diploid cells may also apply to tetraploid
cells.
[0235] Yeast Mating, as used herein, refers broadly to a process by
which two haploid yeast cells naturally fuse to form one diploid
yeast cell.
[0236] Meiosis, as used herein, refers broadly to a process by
which a diploid yeast cell undergoes reductive division to form
four haploid spore products. Each spore may then germinate and form
a haploid vegetatively growing cell line.
[0237] Variable region or VR as used herein refers broadly to the
domains within each pair of light and heavy chains in an antibody
that are involved directly in binding the antibody to the antigen.
Each heavy chain has at one end a variable domain (V.sub.H)
followed by a number of constant domains. Each light chain has a
variable domain (V.sub.L) at one end and a constant domain at its
other end; the constant domain of the light chain is aligned with
the first constant domain of the heavy chain, and the light chain
variable domain is aligned with the variable domain of the heavy
chain.
[0238] Variants, as used herein refers broadly to single-chain
antibodies, dimers, multimers, sequence variants, and domain
substitution variants. Single-chain antibodies such as SMIPs, shark
antibodies, nanobodies (e.g., Camelidiae antibodies). Sequence
variants can be specified by percentage identity (similarity,
sequence homology) e.g., 99%, 95%, 90%, 85%, 80%, 70%, 60%, or by
numbers of permitted conservative or non-conservative
substitutions. Domain substitution variants include replacement of
a domain of one protein with a similar domain of a related protein.
A similar domain may be identified by similarity of sequence,
structure (actual or predicted), or function. For example, domain
substitution variants include the substitution of at least one CDRs
and/or framework regions.
[0239] The techniques and procedures are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification. See,
e.g., Sambrook, et al. (2001) Molec. Cloning: Lab. Manual[3.sup.rd
Ed] Cold Spring Harbor Laboratory Press. Standard techniques may be
used for recombinant DNA, oligonucleotide synthesis, and tissue
culture, and transformation (e.g., electroporation, lipofection).
Enzymatic reactions and purification techniques may be performed
according to manufacturer's specifications or as commonly
accomplished in the art or as described herein. The nomenclatures
utilized in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
may be used for chemical syntheses, chemical analyses,
pharmaceutical preparation, formulation, and delivery, and
treatment of patients.
Anti-IL-6 Antibodies for Treating Psoriatic Arthritis
[0240] The present technology also relates to compositions,
methods, and uses of anti-IL-6 antibodies and/or antigen-binding
fragments thereof according to the subject technology for treating,
preventing, or alleviating the onset of psoriatic arthritis.
[0241] The subject therapy may comprise administering the antibody
prior or concurrent to development of the symptoms of psoriatic
arthritis. Particularly this may be used in patients who have shown
signs of inadequate response to Non-Steroidal Anti-Inflammatory
Drugs (NSAIDs) and/or non-biologic Disease Modifying Anti-Rheumatic
Drugs (DMARDs). Non-biologic DMARDs include, but are not limited
to: Mycophenolate mofetil (CellCept.RTM.), calcineurin inhibitors
(e.g., cyclosporine, sirolimus, everolimus), oral retinoids,
azathioprine, fumeric acid esters, D-penicillamine, and
cyclophosphamide. Non-Steroidal Anti-Inflammatory Drugs (NSAIDs)
including but are not limited to Salicylates (e.g., Aspirin
(acetylsalicylic acid), Diflunisal, Salsalate); Propionic acid
derivatives (e.g., Ibuprofen, Naproxen, Fenoprofen, Ketoprofen,
Flurbiprofen, Oxaprozin, Loxoprofen); Acetic acid derivatives
(e.g., Indomethacin, Sulindac, Etodolac, Ketorolac, Diclofenac,
Nabumetone); Enolic acid (Oxicam) derivatives (e.g., Piroxicam,
Meloxicam, Tenoxicam, Droxicam, Lornoxicam, Isoxicam); Fenamic acid
derivatives (Fenamates) (e.g., Mefenamic acid, Meclofenamic acid,
Flufenamic acid, Tolfenamic acid); Selective COX-2 inhibitors
(Coxibs) (e.g., Celecoxib, Rofecoxib, Valdecoxib, Parecoxib,
Lumiracoxib, Etoricoxib, Firocoxib), Sulphonanilides (e.g.,
Nimesulide), and Licofelone.
[0242] The subject technology provides for method of treating
psoriatic arthritis comprising administration of a composition
comprising an effective amount of an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6,
Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17,
Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28,
Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36 antibody, or an
antigen-binding fragment thereof, to a subject in need thereof,
wherein the antibody, or antigen-binding fragment thereof,
specifically binds to IL-6.
[0243] The subject technology also provides for method of
preventing psoriatic arthritis comprising administration of a
composition comprising an effective amount of an Ab1, Ab2, Ab3,
Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15,
Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,
Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36
antibody, or an antigen-binding fragment thereof, to a subject in
need thereof, wherein the antibody, or antigen-binding fragment
thereof, specifically binds to IL-6.
[0244] In either the methods of treatment or prevention, the
antibody, or antigen-binding fragment thereof, is aglycosylated.
Further, the antibody, or antigen-binding fragment thereof, may
contain an Fc region that has been modified to alter effector
function, half-life, proteolysis, and/or glycosylation.
Additionally, the antibody, or antigen-binding fragment thereof, is
a human, humanized, single chain, or chimeric antibody.
[0245] Further, the method of treating or preventing psoriatic
arthritis may comprise administering a composition comprises at
least about 25, 80, 100, 160, 200, or 320 mg of an Ab1, Ab2, Ab3,
Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15,
Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26,
Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36
antibody, or an antigen-binding fragment thereof. The method of
treating or preventing psoriatic arthritis may comprise
administering a composition comprises at least about 25, 80, 100,
160, 200, or 320 mg of an Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8,
Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19,
Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30,
Ab31, Ab32, Ab33, Ab34, Ab35, or Ab36 antibody, or an
antigen-binding fragment thereof, subcutaneously every 4 weeks for
at least 8, 16, 20, or 24 weeks.
[0246] In an embodiment of the subject technology, IL-6 antagonists
such as Ab1 described herein are useful for ameliorating or
reducing the symptoms of, or treating, or preventing, psoriatic
arthritis. The IL-6 antagonists described herein (e.g., Ab1-Ab36)
is administered in a therapeutically effective amount to patients
in need of treatment of psoriatic arthritis in the form of a
pharmaceutical composition formulated for the treatment of
psoriatic arthritis.
[0247] The dosing regimen is based on pharmacokinetic and
pharmacodynamic data from previous studies. For example, in the
advanced cancer clinical trial, single IV doses of 80, 160, and 320
mg ALD518 decreased CRP levels to normal or near normal for 12
weeks. In the rheumatoid arthritis clinical trial, 2 IV doses of
80, 160, and 320 mg ALD518 given 8 weeks apart decreased CRP levels
to normal or nearly normal for 16 weeks. However, in the NSCLC
clinical trial, CRP levels were decreased 2 weeks after the first
of 3 IV doses of 80, 160, and 320 mg of ALD518, 8 weeks apart, but
increased prior to the second dose. In addition, the elimination
half-life, which is based on free ALD518, was 28 days in the normal
subject, advanced cancer, but was reduced to 21 days in the NSCLC
clinical trial. ALD518* is an asialated, humanized anti-IL-6
monoclonal antibody with a half-life of .about.30 days containing
the humanized variable heavy and light sequences set forth in SEQ
ID NO: 19 and 20. Pharmacokinetic (PK) modeling of data from the
NSCLC clinical trial indicates that doses of ALD518 80 mg
administered once every 3 weeks would not result in trough ALD518
concentrations high enough to fully suppress CRP. Since CRP levels
in the published data in head and neck cancer studies can be as
high as those seen in subjects with NSCLC, the doses may be 160 mg
and 320 mg of humanized monoclonal antibody that selectively binds
IL-6 administered every 4 weeks. See, e.g., Gallo, et al. (1992) Br
J Cancer 65:479-80; Duffy, et al. (2008) Cancer 113:750-7.
Examplary ALD518 plasma concentration effective to inhibit CRP may
be at least about 15 .mu.g/mL.
[0248] ALD518 is an exemplary humanized anti-IL-6 monoclonal
antibody. ALD518 may be supplied as a pH 6.0 frozen injection in
single-use vials (e.g., 80, 160, or 320 mg) for intravenous
administration. In the 80 mg dose, exemplary non-active excipients
include but are not limited to 25 mM histidine and 250 mM sorbitol.
In the 160 mg formulation, exemplary non-active excipients include
but are not limited to 25 mM histidine, 250 mM sorbitol, and 0.015%
polysorbate 80. Compositions comprising humanized monoclonal
antibodies that selectively bind IL-6 (e.g., ALD518) may be
sterile, preservative-free frozen liquid injection in depyrogenated
sterile vials, which are stoppered and sealed containing
approximately 80 mg (e.g., 7.6 mL in a 10 mLvial) or approximately
160 mg (e.g., 4 mL in a 5 mL vial). For example, one dose of ALD518
(e.g., 160 mg or 320 mg) in 250 mL 0.9% saline may be administered
IV over a period of at least about one hour (.+-.15 minutes) on the
morning of RT Day 1 and RT Treatment Week 4.
[0249] In one embodiment of the subject technology, IL-6
antagonists described herein (e.g., Ab1) are useful for
ameliorating or reducing the symptoms of, or treating, or
preventing psoriatic arthritis.
[0250] In another embodiment of the subject technology, IL-6
antagonists described herein are administered to a patient in
combination with another active agent. For example, an IL-6
antagonist such as Ab1 may be co-administered with at least one
chemotherapy agents, such as VEGF antagonists, EGFR antagonists,
platins, taxols, irinotecan, 5-fluorouracil, gemcytabine,
leucovorine, steroids, cyclophosphamide, melphalan, vinca alkaloids
(e.g., vinblastine, vincristine, vindesine and vinorelbine),
mustines, tyrosine kinase inhibitors, radiotherapy, sex hormone
antagonists, selective androgen receptor modulators, selective
estrogen receptor modulators, PDGF antagonists, TNF antagonists,
IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12R
antagonists, Erbitux.RTM. (cetuximab), Avastin.RTM. (bevacizumab),
Pertuzumab, anti-CD20 antibodies, Rituxan.RTM. (rituximab),
ocrelizumab, ofatumumab, DXL625, Herceptin.RTM. (trastuzumab), or
any combination thereof.
Anti-IL-6 Antibodies and Binding Fragments Thereof
[0251] The subject technology includes antibodies having binding
specificity to IL-6 and possessing a variable light chain sequence
comprising the sequence set forth in the polypeptide sequence of
SEQ ID NO: 2 or SEQ ID NO: 709 and humanized versions and variants
thereof including those set forth in FIGS. 1 and 8-11, and those
identified in Table 1.
[0252] Antibodies consist of two identical light polypeptide chains
of molecular weight approximately 23,000 daltons (the "light
chain"), and two identical heavy chains of molecular weight
53,000-70,000 (the "heavy chain"). The four chains are joined by
disulfide bonds in a "Y" configuration wherein the light chains
bracket the heavy chains starting at the mouth of the "Y"
configuration. The "branch" portion of the "Y" configuration is
designated the Fab region; the stem portion of the "Y"
configuration is designated the Fc region. The amino acid sequence
orientation runs from the N-terminal end at the top of the "Y"
configuration to the C-terminal end at the bottom of each chain.
The N-terminal end possesses the variable region having specificity
for the antigen that elicited it, and is approximately 100 amino
acids in length, there being slight variations between light and
heavy chain and from antibody to antibody.
[0253] The variable region is linked in each chain to a constant
region that extends the remaining length of the chain and that
within a particular class of antibody does not vary with the
specificity of the antibody (i.e., the antigen eliciting it). There
are five known major classes of constant regions that determine the
class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE
corresponding to .gamma., .mu., .alpha., .delta., and .epsilon.
(gamma, mu, alpha, delta, or epsilon) heavy chain constant
regions). The constant region or class determines subsequent
effector function of the antibody, including activation of
complement (Kabat, E. A. (1976) Structural Concepts in Immunology
and Immunochemistry, 2nd Ed., pp. 413-436, Holt, Rinehart,
Winston), and other cellular responses (Andrews, et al. (1980)
Clinical Immunobiology pp 1-18, W. B. Sanders; Kohl, et al. (1983)
Immunology 48: 187); while the variable region determines the
antigen with which it will react. Light chains are classified as
either .kappa. (kappa) or .lamda. (lambda). Each heavy chain class
can be paired with either kappa or lambda light chain. The light
and heavy chains are covalently bonded to each other, and the
"tail" portions of the two heavy chains are bonded to each other by
covalent disulfide linkages when the immunoglobulins are generated
either by hybridomas or by B cells.
[0254] For example, antibodies or antigen binding fragments or
variants thereof may be produced by genetic engineering. In this
technique, as with other methods, antibody-producing cells are
sensitized to the desired antigen or immunogen. The messenger RNA
isolated from antibody producing cells is used as a template to
make cDNA using PCR amplification. A library of vectors, each
containing one heavy chain gene and one light chain gene retaining
the initial antigen specificity, is produced by insertion of
appropriate sections of the amplified immunoglobulin cDNA into the
expression vectors. A combinatorial library is constructed by
combining the heavy chain gene library with the light chain gene
library. This results in a library of clones which co-express a
heavy and light chain (resembling the Fab fragment or antigen
binding fragment of an antibody molecule). The vectors that carry
these genes are co-transfected into a host cell. When antibody gene
synthesis is induced in the transfected host, the heavy and light
chain proteins self-assemble to produce active antibodies that can
be detected by screening with the antigen or immunogen.
[0255] Antibody coding sequences of interest include those encoded
by native sequences, as well as nucleic acids that, by virtue of
the degeneracy of the genetic code, are not identical in sequence
to the disclosed nucleic acids, and variants thereof. Variant
polypeptides can include amino acid (aa) substitutions, additions
or deletions. The amino acid substitutions can be conservative
amino acid substitutions or substitutions to eliminate
non-essential amino acids, such as to alter a glycosylation site,
or to minimize misfolding by substitution or deletion of at least
one cysteine residues that are not necessary for function. Variants
can be designed so as to retain or have enhanced biological
activity of a particular region of the protein (e.g., a functional
domain, catalytic amino acid residues). Variants also include
fragments of the polypeptides disclosed herein, particularly
biologically active fragments and/or fragments corresponding to
functional domains. Techniques for in vitro mutagenesis of cloned
genes are known. Also included in the subject technology are
polypeptides that have been modified using ordinary molecular
biological techniques so as to improve their resistance to
proteolytic degradation or to optimize solubility properties or to
render them more suitable as a therapeutic agent.
[0256] Chimeric antibodies may be made by recombinant means by
combining the variable light and heavy chain regions (V.sub.L and
V.sub.H), obtained from antibody producing cells of one species
with the constant light and heavy chain regions from another.
Typically chimeric antibodies utilize rodent or rabbit variable
regions and human constant regions, in order to produce an antibody
with predominantly human domains. The production of such chimeric
antibodies is well known in the art, and may be achieved by
standard means (as described, e.g., in U.S. Pat. No. 5,624,659,
incorporated herein by reference in its entirety). It is further
contemplated that the human constant regions of chimeric antibodies
of the subject technology may be selected from IgG1, IgG2, IgG3,
IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13,
IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19 constant regions.
[0257] Humanized antibodies are engineered to contain even more
human-like immunoglobulin domains, and incorporate only the
complementarity-determining regions of the animal-derived antibody.
This is accomplished by carefully examining the sequence of the
hyper-variable loops of the variable regions of the monoclonal
antibody, and fitting them to the structure of the human antibody
chains. Although facially complex, the process is straightforward
in practice. See, e.g., U.S. Pat. No. 6,187,287. In a preferred
embodiment, humanization may be effected as disclosed in detail
infra. This scheme grafts CDRs onto human FRs highly homologous to
the parent antibody that is being humanized.
[0258] Immunoglobulins and fragments thereof may be modified
post-translationally, e.g. to add effector moieties such as
chemical linkers, detectable moieties, such as fluorescent dyes,
enzymes, toxins, substrates, bioluminescent materials, radioactive
materials, chemiluminescent moieties and the like, or specific
binding moieties, such as streptavidin, avidin, or biotin, and the
like may be utilized in the methods and compositions of the present
subject technology. Examples of additional effector molecules are
provided infra.
[0259] The subject technology also includes antibodies having
binding specificity to IL-6 and possessing a variable heavy chain
sequence comprising the sequence set forth in the polypeptide
sequences of SEQ ID NO: 3 and SEQ ID NO: 657 and humanized versions
and variants thereof including those set forth in FIGS. 1 and 8-11,
and those identified in Table 1.
[0260] The subject technology further includes antibodies having
binding specificity to IL-6 and possessing a variable heavy chain
sequence which is a modified version of SEQ ID NO: 3 wherein the
tryptophan residue in CDR2 is changed to a serine as set forth in
the polypeptide sequence of SEQ ID NO: 658 and humanized versions
and variants thereof including those set forth in FIGS. 1 and 8-11,
and those identified in Table 1.
[0261] The subject technology further contemplates antibodies
comprising at least one of the polypeptide sequences of SEQ ID NO:
4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NOs: 2 or
709, and/or at least one of the polypeptide sequences of SEQ ID NO:
7; SEQ ID NO: 8 or 120; and SEQ ID NO: 9 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable heavy chain sequence of SEQ ID NOs: 3 or
19 or 657, or combinations of these polypeptide sequences. In
another embodiment of the subject technology, the antibodies of the
subject technology include combinations of the CDRs and the
variable heavy and light chain sequences set forth above.
[0262] In another embodiment, the subject technology contemplates
other antibodies, such as for example chimeric antibodies,
comprising at least one of the polypeptide sequences of SEQ ID NO:
4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NOs: 2 or
709, and/or at least one of the polypeptide sequences of SEQ ID NO:
7; SEQ ID NO: 8 or 120; and SEQ ID NO: 9 which correspond to the
complementarity-determining regions (CDRs, or hypervariable
regions) of the variable heavy chain sequence of SEQ ID NOs: 3 or
19 or 657, or combinations of these polypeptide sequences. In
another embodiment of the subject technology, the antibodies of the
subject technology include combinations of the CDRs and humanized
versions of the variable heavy and light chain sequences set forth
above.
[0263] The subject technology also contemplates fragments of the
antibody having binding specificity to IL-6. In one embodiment of
the subject technology, antibody fragments of the subject
technology comprise, or alternatively consist of, humanized
versions of the polypeptide sequence of SEQ ID NO: 2, 20, 647, 651,
660, 666, 699, 702, 706, or 709. In another embodiment of the
subject technology, antibody fragments of the subject technology
comprise, or alternatively consist of, humanized versions of the
polypeptide sequence of SEQ ID NO: 3, 18, 19, 652, 656, 657, 658,
661, 664, 665, 704, or 708.
[0264] In a further embodiment of the subject technology, fragments
of the antibody having binding specificity to IL-6 comprise, or
alternatively consist of, at least one of the polypeptide sequences
of SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to
the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 2 or
SEQ ID NO: 709.
[0265] In a further embodiment of the subject technology, fragments
of the antibody having binding specificity to IL-6 comprise, or
alternatively consist of, at least one of the polypeptide sequences
of SEQ ID NO: 7; SEQ ID NO: 8 or SEQ ID NO: 120; and SEQ ID NO: 9
which correspond to the complementarity-determining regions (CDRs,
or hypervariable regions) of the variable heavy chain sequence of
SEQ ID NO: 3 or 657 or 19.
[0266] The subject technology also contemplates antibody fragments
which include at least one of the antibody fragments described
herein. In one embodiment of the subject technology, fragments of
the antibodies having binding specificity to IL-6 comprise, or
alternatively consist of, one, two, three or more, including all of
the following antibody fragments: the variable light chain region
of SEQ ID NO: 2; the variable heavy chain region of SEQ ID NO: 3;
the complementarity-determining regions (SEQ ID NO: 4; SEQ ID NO:
5; and SEQ ID NO: 6) of the variable light chain region of SEQ ID
NOs: 2 or 709; and the complementarity-determining regions (SEQ ID
NO: 7; SEQ ID NO: 8 or SEQ ID NO: 120; and SEQ ID NO: 9) of the
variable heavy chain region of SEQ ID NOs: 3 or 657 or 19.
[0267] The subject technology also contemplates variants wherein
either of the heavy chain polypeptide sequences of SEQ ID NO: 18 or
SEQ ID NO: 19 is substituted for the heavy chain polypeptide
sequence of SEQ ID NOs: 3 or 657; the light chain polypeptide
sequence of SEQ ID NO: 20 is substituted for the light chain
polypeptide sequence of SEQ ID NO: 2 or SEQ ID NO: 709; and the
heavy chain CDR sequence of SEQ ID NO: 120 is substituted for the
heavy chain CDR sequence of SEQ ID NO: 8.
[0268] In a preferred embodiment of the subject technology, the
anti-IL-6 antibody is Ab1, comprising SEQ ID NO: 2 and SEQ ID NO:
3, or more particularly an antibody comprising SEQ ID NO: 657 and
SEQ ID NO: 709 (which are respectively encoded by the nucleic acid
sequences in SEQ ID NO: 700 and SEQ ID NO: 723) or one comprised of
the alternative SEQ ID NOs set forth in the preceding paragraph,
and having at least one of the biological activities set forth
herein. In a preferred embodiment the anti-IL-6 antibody will
comprise a humanized sequence as shown in FIGS. 8-11.
[0269] Sequences of anti-IL-6 antibodies of the present subject
technology are shown in Table 1. Exemplary sequence variants other
alternative forms of the heavy and light chains of Ab1 through Ab7
are shown. The antibodies of the present subject technology
encompass additional sequence variants, including conservative
substitutions, substitution of at least one CDR sequences and/or FR
sequences.
[0270] Exemplary Ab1 embodiments include an antibody comprising a
variant of the light chain and/or heavy chain. Exemplary variants
of the light chain of Ab1 include the sequence of any of the Ab1
light chains shown (i.e., any of SEQ ID NO: 2, 20, 647, 651, 660,
666, 699, 702, 706, or 709) wherein the entire CDR1 sequence is
replaced or wherein at least one residues in the CDR1 sequence is
substituted by the residue in the corresponding position of any of
the other light chain CDR1 sequences set forth (i.e., any of SEQ ID
NO: 23, 39, 55, 71, 87, 103, 124, 140, 156, 172, 188, 204, 220,
236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428,
444, 460, 476, 492, 508, 524, 540, 556, or 572); and/or wherein the
entire CDR2 sequence is replaced or wherein at least one residues
in the CDR2 sequence is substituted by the residue in the
corresponding position of any of the other light chain CDR2
sequences set forth (i.e., any of SEQ ID NOs: 24, 40, 56, 72, 88,
104, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301,
317, 333, 349, 365, 381, 397, 413, 429, 445, 461, 477, 493, 509,
525, 541, 557, or 573); and/or wherein the entire CDR3 sequence is
replaced or wherein at least one residues in the CDR3 sequence is
substituted by the residue in the corresponding position of any of
the other light chain CDR3 sequences set forth (i.e., any of SEQ ID
NOs: 25, 41, 57, 73, 89, 105, 126, 142, 158, 174, 190, 206, 222,
238, 254, 270, 286, 302, 318, 334, 350, 366, 382, 398, 414, 430,
446, 462, 478, 494, 510, 526, 542, 558, or 574).
[0271] Exemplary variants of the heavy chain of Ab1 include the
sequence of any of the Ab1 heavy chains shown (i.e., any of SEQ ID
NO: 3, 18, 19, 652, 656, 657, 658, 661, 664, 665, 704, or 708)
wherein the entire CDR1 sequence is replaced or wherein at least
one residues in the CDR1 sequence is substituted by the residue in
the corresponding position of any of the other heavy chain CDR1
sequences set forth (i.e., any of SEQ ID NO: 26, 42, 58, 74, 90,
106, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303,
319, 335, 351, 367, 383, 399, 415, 431, 447, 463, 479, 495, 511,
527, 543, 559, or 575); and/or wherein the entire CDR2 sequence is
replaced or wherein at least one residues in the CDR2 sequence is
substituted by the residue in the corresponding position of an Ab1
heavy chain CDR2, such as those set forth in Table 1 (i.e., any of
SEQ ID NO: 8, or 120) or any of the other heavy chain CDR2
sequences set forth (i.e., any of SEQ ID NO: 27, 43, 59, 75, 91,
107, 121, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288,
304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496,
512, 528, 544, 560, or 576); and/or wherein the entire CDR3
sequence is replaced or wherein at least one residues in the CDR3
sequence is substituted by the residue in the corresponding
position of any of the other heavy chain CDR3 sequences set forth
(i.e., any of SEQ ID NO: 28, 44, 60, 76, 92, 108, 129, 145, 161,
177, 193, 209, 225, 241, 257, 273, 289, 305, 321, 337, 353, 369,
385, 401, 417, 433, 449, 465, 481, 497, 513, 529, 545, 561, or
577).
[0272] In another embodiment, the subject technology contemplates
other antibodies, such as for example chimeric or humanized
antibodies, comprising at least one of the polypeptide sequences of
SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to
the complementarity-determining regions (CDRs, or hypervariable
regions) of the variable light chain sequence of SEQ ID NO: 2,
and/or at least one of the polypeptide sequences of SEQ ID NO: 7
(CDR1); SEQ ID NO: 8 (CDR2); SEQ ID NO: 120 (CDR2); and SEQ ID NO:
9 (CDR3) which correspond to the complementarity-determining
regions (CDRs, or hypervariable regions) of the variable heavy
chain sequence of SEQ ID NO: 3 or SEQ ID NO: 19, or combinations of
these polypeptide sequences. In another embodiment of the subject
technology, the antibodies of the subject technology include
combinations of the CDRs and the variable heavy and light chain
sequences set forth above including those set forth in FIGS. 1 and
8-11, and those identified in Table 1.
[0273] In another embodiment the anti-IL-6 antibody of the subject
technology is one comprising at least one of the following: a CDR1
light chain encoded by the sequence in SEQ ID NO: 12 or SEQ ID NO:
694; a light chain CDR2 encoded by the sequence in SEQ ID NO: 13; a
light chain CDR3 encoded by the sequence in SEQ ID NO: 14 or SEQ ID
NO: 695; a heavy chain CDR1 encoded by the sequence in SEQ ID NO:
15, a heavy chain CDR2 encoded by SEQ ID NO: 16 or SEQ ID NO: 696
and a heavy chain CDR3 encoded by SEQ ID NO: 17 or SEQ ID NO: 697.
In addition the subject technology embraces such nucleic acid
sequences and variants thereof.
[0274] In another embodiment the subject technology is directed to
amino acid sequences corresponding to the CDRs of said anti-IL-6
antibody which are selected from SEQ ID NO: 4 (CDR1), SEQ ID NO: 5
(CDR2), SEQ ID NO: 6 (CDR3), SEQ ID NO: 7, SEQ ID NO: 120 and SEQ
ID NO: 9.
[0275] In another embodiment the anti-IL-6 antibody of the subject
technology comprises a light chain nucleic acid sequence of SEQ ID
NO: 10, 662, 698, 701, 705, 720, 721, 722, or 723; and/or a heavy
chain nucleic acid sequence of SEQ ID NO: 11, 663, 700, 703, 707,
724, or 725. In addition the subject technology is directed to the
corresponding polypeptides encoded by any of the foregoing nucleic
acid sequences and combinations thereof.
[0276] In a specific embodiment of the subject technology the
anti-IL-6 antibodies or a portion thereof will be encoded by a
nucleic acid sequence selected from those comprised in SEQ ID NO:
10, 12, 13, 14, 662, 694, 695, 698, 701, 705, 720, 721, 722, 723,
11, 15, 16, 17, 663, 696, 697, 700, 703, 707, 724, and 725. For
example the CDR1 in the light chain may be encoded by SEQ ID NO: 12
or 694, the CDR2 in the light chain may be encoded by SEQ ID NO:
13, the CDR3 in the light chain may be encoded by SEQ ID NO: 14 or
695; the CDR1 in the heavy chain may be encoded by SEQ ID NO: 15,
the CDR2 in the heavy chain may be encoded by SEQ ID NO: 16 or 696,
the CDR3 in the heavy chain may be encoded by SEQ ID NO: 17 or 697.
As discussed infra antibodies containing these CDRs may be
constructed using appropriate human frameworks based on the
humanization methods disclosed herein.
[0277] In another specific embodiment of the subject technology the
variable light chain will be encoded by SEQ ID NO: 10, 662, 698,
701, 705, 720, 721, 722, or 723 and the variable heavy chain of the
anti-IL-6 antibodies will be encoded by SEQ ID NO: 11, 663, 700,
703, 707, 724, or 725.
[0278] In a more specific embodiment variable light and heavy
chains of the anti-IL-6 antibody respectively will be encoded by
SEQ ID NO: 10 and 11, or SEQ ID NO: 698 and SEQ ID NO: 700 or SEQ
ID NO: 701 and SEQ ID NO: 703 or SEQ ID NO: 705 and SEQ ID NO:
707.
[0279] In another specific embodiment the subject technology covers
nucleic acid constructs containing any of the foregoing nucleic
acid sequences and combinations thereof as well as recombinant
cells containing these nucleic acid sequences and constructs
containing wherein these nucleic acid sequences or constructs may
be extrachromosomal or integrated into the host cell genome.
[0280] In another specific embodiment the subject technology covers
polypeptides containing any of the CDRs or combinations thereof
recited in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,
SEQ ID NO: 8, SEQ ID NO: 120, SEQ ID NO: 9 or polypeptides
comprising any of the variable light polypeptides comprised in SEQ
ID NO: 2, 20, 647, 651, 660, 666, 699, 702, 706, or 709 and/or the
variable heavy polypeptides comprised in SEQ ID NO: 3, 18, 19, 652,
656, 657, 658, 661, 664, 665, 704, or 708. These polypeptides
optionally may be attached directly or indirectly to other
immunoglobulin polypeptides or effector moieties such as
therapeutic or detectable entities.
[0281] In another embodiment the anti-IL-6 antibody is one
comprising at least one of the following: a variable light chain
encoded by the sequence in SEQ ID NO: 10 or SEQ ID NO: 698 or SEQ
ID NO: 701 or SEQ ID NO: 705 and a variable chain encoded by the
sequence in SEQ ID NO: 11 or SEQ ID NO: 700 or SEQ ID NO: 703 or
SEQ ID NO: 707.
[0282] In another embodiment the anti-IL-6 antibody is a variant of
the foregoing sequences that includes at least one substitution in
the framework and/or CDR sequences and which has at least one of
the properties of Ab1 in vitro and/or upon in vivo
administration.
[0283] These in vitro and in vivo properties are described in more
detail in the examples below and include: competing with Ab1 for
binding to IL-6 and/or peptides thereof; having a binding affinity
(Kd) for IL-6 of less than about 50 picomolar, and/or a rate of
dissociation (K.sub.off) from IL-6 of less than or equal to
10.sup.-4 S.sup.-1; having an in-vivo half-life of at least about
22 days in a healthy human subject; ability to prevent or treat
hypoalbunemia; ability to prevent or treat elevated CRP; ability to
prevent or treat abnormal coagulation; and/or ability to decrease
the risk of thrombosis in an individual having a disease or
condition associated with increased risk of thrombosis. Additional
non-limiting examples of anti-IL-6 activity are set forth herein,
for example, under the heading "Anti-IL-6 Activity."
[0284] In another embodiment the anti-IL-6 antibody includes at
least one of the Ab1 light-chain and/or heavy chain CDR sequences
(see Table 1) or variant(s) thereof which has at least one of the
properties of Ab1 in vitro and/or upon in vivo administration
(examples of such properties are discussed in the preceding
paragraph). One of skill in the art would understand how to combine
these CDR sequences to form an antigen-binding surface, e.g. by
linkage to at least one scaffold which may comprise human or other
mammalian framework sequences, or their functional orthologs
derived from a SMIP, camelbody, nanobody, IgNAR or other
immunoglobulin or other engineered antibody. For example,
embodiments may specifically bind to human IL-6 and include one,
two, three, four, five, six, or more of the following CDR sequences
or variants thereof: a polypeptide having at least 72.7% (i.e., 8
out of 11 amino acids) identity to the light chain CDR1 of SEQ ID
NO: 4; a polypeptide having at least 81.8% (i.e., 9 out of 11 amino
acids) identity to the light chain CDR1 of SEQ ID NO: 4; a
polypeptide having at least 90.9% (i.e., 10 out of 11 amino acids)
identity to the light chain CDR1 of SEQ ID NO: 4; a polypeptide
having 100% (i.e., 11 out of 11 amino acids) identity to the light
chain CDR1 of SEQ ID NO: 4; a polypeptide having at least 85.7%
(i.e., 6 out of 7 amino acids) identity to the light chain CDR2 of
SEQ ID NO: 5; a polypeptide having 100% (i.e., 7 out of 7 amino
acids) identity to the light chain CDR2 of SEQ ID NO: 5; a
polypeptide having at least 50% (i.e., 6 out of 12 amino acids)
identity to the light chain CDR3 of SEQ ID NO: 6; a polypeptide
having at least 58.3% (i.e., 7 out of 12 amino acids) identity to
the light chain CDR3 of SEQ ID NO: 6;
[0285] a polypeptide having at least 66.6% (i.e., 8 out of 12 amino
acids) identity to the light chain CDR3 of SEQ ID NO: 6; a
polypeptide having at least 75% (i.e., 9 out of 12 amino acids)
identity to the light chain CDR3 of SEQ ID NO: 6; a polypeptide
having at least 83.3% (i.e., 10 out of 12 amino acids) identity to
the light chain CDR3 of SEQ ID NO: 6; a polypeptide having at least
91.6% (i.e., 11 out of 12 amino acids) identity to the light chain
CDR3 of SEQ ID NO: 6; a polypeptide having 100% (i.e., 12 out of 12
amino acids) identity to the light chain CDR3 of SEQ ID NO: 6; a
polypeptide having at least 80% (i.e., 4 out of 5 amino acids)
identity to the heavy chain CDR1 of SEQ ID NO: 7; a polypeptide
having 100% (i.e., 5 out of 5 amino acids) identity to the heavy
chain CDR1 of SEQ ID NO: 7; a polypeptide having at least 50%
(i.e., 8 out of 16 amino acids) identity to the heavy chain CDR2 of
SEQ ID NO: 120; a polypeptide having at least 56.2% (i.e., 9 out of
16 amino acids) identity to the heavy chain CDR2 of SEQ ID NO: 120;
a polypeptide having at least 62.5% (i.e., 10 out of 16 amino
acids) identity to the heavy chain CDR2 of SEQ ID NO: 120; a
polypeptide having at least 68.7% (i.e., 11 out of 16 amino acids)
identity to the heavy chain CDR2 of SEQ ID NO: 120; a polypeptide
having at least 75% (i.e., 12 out of 16 amino acids) identity to
the heavy chain CDR2 of SEQ ID NO: 120;
[0286] a polypeptide having at least 81.2% (i.e., 13 out of 16
amino acids) identity to the heavy chain CDR2 of SEQ ID NO: 120; a
polypeptide having at least 87.5% (i.e., 14 out of 16 amino acids)
identity to the heavy chain CDR2 of SEQ ID NO: 120; a polypeptide
having at least 93.7% (i.e., 15 out of 16 amino acids) identity to
the heavy chain CDR2 of SEQ ID NO: 120; a polypeptide having 100%
(i.e., 16 out of 16 amino acids) identity to the heavy chain CDR2
of SEQ ID NO: 120; a polypeptide having at least 33.3% (i.e., 4 out
of 12 amino acids) identity to the heavy chain CDR3 of SEQ ID NO:
9; a polypeptide having at least 41.6% (i.e., 5 out of 12 amino
acids) identity to the heavy chain CDR3 of SEQ ID NO: 9; a
polypeptide having at least 50% (i.e., 6 out of 12 amino acids)
identity to the heavy chain CDR3 of SEQ ID NO: 9; a polypeptide
having at least 58.3% (i.e., 7 out of 12 amino acids) identity to
the heavy chain CDR3 of SEQ ID NO: 9; a polypeptide having at least
66.6% (i.e., 8 out of 12 amino acids) identity to the heavy chain
CDR3 of SEQ ID NO: 9; a polypeptide having at least 75% (i.e., 9
out of 12 amino acids) identity to the heavy chain CDR3 of SEQ ID
NO: 9; a polypeptide having at least 83.3% (i.e., 10 out of 12
amino acids) identity to the heavy chain CDR3 of SEQ ID NO: 9; a
polypeptide having at least 91.6% (i.e., 11 out of 12 amino acids)
identity to the heavy chain CDR3 of SEQ ID NO: 9; a polypeptide
having 100% (i.e., 12 out of 12 amino acids) identity to the heavy
chain CDR3 of SEQ ID NO: 9; a polypeptide having at least 90.9%
(i.e., 10 out of 11 amino acids) similarity to the light chain CDR1
of SEQ ID NO: 4; a polypeptide having 100% (i.e., 11 out of 11
amino acids) similarity to the light chain CDR1 of SEQ ID NO: 4; a
polypeptide having at least 85.7% (i.e., 6 out of 7 amino acids)
similarity to the light chain CDR2 of SEQ ID NO: 5; a polypeptide
having 100% (i.e., 7 out of 7 amino acids) similarity to the light
chain CDR2 of SEQ ID NO: 5; a polypeptide having at least 66.6%
(i.e., 8 out of 12 amino acids) similarity to the light chain CDR3
of SEQ ID NO: 6; a polypeptide having at least 75% (i.e., 9 out of
12 amino acids) similarity to the light chain CDR3 of SEQ ID NO: 6;
a polypeptide having at least 83.3% (i.e., 10 out of 12 amino
acids) similarity to the light chain CDR3 of SEQ ID NO: 6; a
polypeptide having at least 91.6% (i.e., 11 out of 12 amino acids)
similarity to the light chain CDR3 of SEQ ID NO: 6; a polypeptide
having 100% (i.e., 12 out of 12 amino acids) similarity to the
light chain CDR3 of SEQ ID NO: 6; a polypeptide having at least 80%
(i.e., 4 out of 5 amino acids) similarity to the heavy chain CDR1
of SEQ ID NO: 7; a polypeptide having 100% (i.e., 5 out of 5 amino
acids) similarity to the heavy chain CDR1 of SEQ ID NO: 7; a
polypeptide having at least 56.2% (i.e., 9 out of 16 amino acids)
similarity to the heavy chain CDR2 of SEQ ID NO: 120; a polypeptide
having at least 62.5% (i.e., 10 out of 16 amino acids) similarity
to the heavy chain CDR2 of SEQ ID NO: 120; a polypeptide having at
least 68.7% (i.e., 11 out of 16 amino acids) similarity to the
heavy chain CDR2 of SEQ ID NO: 120; a polypeptide having at least
75% (i.e., 12 out of 16 amino acids) similarity to the heavy chain
CDR2 of SEQ ID NO: 120; a polypeptide having at least 81.2% (i.e.,
13 out of 16 amino acids) similarity to the heavy chain CDR2 of SEQ
ID NO: 120; a polypeptide having at least 87.5% (i.e., 14 out of 16
amino acids) similarity to the heavy chain CDR2 of SEQ ID NO: 120;
a polypeptide having at least 93.7% (i.e., 15 out of 16 amino
acids) similarity to the heavy chain CDR2 of SEQ ID NO: 120; a
polypeptide having 100% (i.e., 16 out of 16 amino acids) similarity
to the heavy chain CDR2 of SEQ ID NO: 120; a polypeptide having at
least 50% (i.e., 6 out of 12 amino acids) similarity to the heavy
chain CDR3 of SEQ ID NO: 9; a polypeptide having at least 58.3%
(i.e., 7 out of 12 amino acids) similarity to the heavy chain CDR3
of SEQ ID NO: 9; a polypeptide having at least 66.6% (i.e., 8 out
of 12 amino acids) similarity to the heavy chain CDR3 of SEQ ID NO:
9; a polypeptide having at least 75% (i.e., 9 out of 12 amino
acids) similarity to the heavy chain CDR3 of SEQ ID NO: 9; a
polypeptide having at least 83.3% (i.e., 10 out of 12 amino acids)
similarity to the heavy chain CDR3 of SEQ ID NO: 9; a polypeptide
having at least 91.6% (i.e., 11 out of 12 amino acids) similarity
to the heavy chain CDR3 of SEQ ID NO: 9; or a polypeptide having
100% (i.e., 12 out of 12 amino acids) similarity to the heavy chain
CDR3 of SEQ ID NO: 9.
[0287] Other exemplary embodiments include at least one
polynucleotides encoding any of the foregoing, e.g., a
polynucleotide encoding a polypeptide that specifically binds to
human IL-6 and includes one, two, three, four, five, six, or more
of the following CDRs or variants thereof:
[0288] a polynucleotide encoding a polypeptide having at least
72.7% (i.e., 8 out of 11 amino acids) identity to the light chain
CDR1 of SEQ ID NO: 4; a polynucleotide encoding a polypeptide
having at least 81.8% (i.e., 9 out of 11 amino acids) identity to
the light chain CDR1 of SEQ ID NO: 4; a polynucleotide encoding a
polypeptide having at least 90.9% (i.e., 10 out of 11 amino acids)
identity to the light chain CDR1 of SEQ ID NO: 4; a polynucleotide
encoding a polypeptide having 100% (i.e., 11 out of 11 amino acids)
identity to the light chain CDR1 of SEQ ID NO: 4; a polynucleotide
encoding a polypeptide having at least 85.7% (i.e., 6 out of 7
amino acids) identity to the light chain CDR2 of SEQ ID NO: 5; a
polynucleotide encoding a polypeptide having 100% (i.e., 7 out of 7
amino acids) identity to the light chain CDR2 of SEQ ID NO: 5; a
polynucleotide encoding a polypeptide having at least 50% (i.e., 6
out of 12 amino acids) identity to the light chain CDR3 of SEQ ID
NO: 6; a polynucleotide encoding a polypeptide having at least
58.3% (i.e., 7 out of 12 amino acids) identity to the light chain
CDR3 of SEQ ID NO: 6; a polynucleotide encoding a polypeptide
having at least 66.6% (i.e., 8 out of 12 amino acids) identity to
the light chain CDR3 of SEQ ID NO: 6; a polynucleotide encoding a
polypeptide having at least 75% (i.e., 9 out of 12 amino acids)
identity to the light chain CDR3 of SEQ ID NO: 6; a polynucleotide
encoding a polypeptide having at least 83.3% (i.e., 10 out of 12
amino acids) identity to the light chain CDR3 of SEQ ID NO: 6; a
polynucleotide encoding a polypeptide having at least 91.6% (i.e.,
11 out of 12 amino acids) identity to the light chain CDR3 of SEQ
ID NO: 6; a polynucleotide encoding a polypeptide having 100%
(i.e., 12 out of 12 amino acids) identity to the light chain CDR3
of SEQ ID NO: 6; a polynucleotide encoding a polypeptide having at
least 80% (i.e., 4 out of 5 amino acids) identity to the heavy
chain CDR1 of SEQ ID NO: 7; a polynucleotide encoding a polypeptide
having 100% (i.e., 5 out of 5 amino acids) identity to the heavy
chain CDR1 of SEQ ID NO: 7; a polynucleotide encoding a polypeptide
having at least 50% (i.e., 8 out of 16 amino acids) identity to the
heavy chain CDR2 of SEQ ID NO: 120; a polynucleotide encoding a
polypeptide having at least 56.2% (i.e., 9 out of 16 amino acids)
identity to the heavy chain CDR2 of SEQ ID NO: 120; a
polynucleotide encoding a polypeptide having at least 62.5% (i.e.,
10 out of 16 amino acids) identity to the heavy chain CDR2 of SEQ
ID NO: 120; a polynucleotide encoding a polypeptide having at least
68.7% (i.e., 11 out of 16 amino acids) identity to the heavy chain
CDR2 of SEQ ID NO: 120; a polynucleotide encoding a polypeptide
having at least 75% (i.e., 12 out of 16 amino acids) identity to
the heavy chain CDR2 of SEQ ID NO: 120; a polynucleotide encoding a
polypeptide having at least 81.2% (i.e., 13 out of 16 amino acids)
identity to the heavy chain CDR2 of SEQ ID NO: 120; a
polynucleotide encoding a polypeptide having at least 87.5% (i.e.,
14 out of 16 amino acids) identity to the heavy chain CDR2 of SEQ
ID NO: 120; a polynucleotide encoding a polypeptide having at least
93.7% (i.e., 15 out of 16 amino acids) identity to the heavy chain
CDR2 of SEQ ID NO: 120; a polynucleotide encoding a polypeptide
having 100% (i.e., 16 out of 16 amino acids) identity to the heavy
chain CDR2 of SEQ ID NO: 120; a polynucleotide encoding a
polypeptide having at least 33.3% (i.e., 4 out of 12 amino acids)
identity to the heavy chain CDR3 of SEQ ID NO: 9; a polynucleotide
encoding a polypeptide having at least 41.6% (i.e., 5 out of 12
amino acids) identity to the heavy chain CDR3 of SEQ ID NO: 9; a
polynucleotide encoding a polypeptide having at least 50% (i.e., 6
out of 12 amino acids) identity to the heavy chain CDR3 of SEQ ID
NO: 9; a polynucleotide encoding a polypeptide having at least
58.3% (i.e., 7 out of 12 amino acids) identity to the heavy chain
CDR3 of SEQ ID NO: 9; a polynucleotide encoding a polypeptide
having at least 66.6% (i.e., 8 out of 12 amino acids) identity to
the heavy chain CDR3 of SEQ ID NO: 9; a polynucleotide encoding a
polypeptide having at least 75% (i.e., 9 out of 12 amino acids)
identity to the heavy chain CDR3 of SEQ ID NO: 9; a polynucleotide
encoding a polypeptide having at least 83.3% (i.e., 10 out of 12
amino acids) identity to the heavy chain CDR3 of SEQ ID NO: 9; a
polynucleotide encoding a polypeptide having at least 91.6% (i.e.,
11 out of 12 amino acids) identity to the heavy chain CDR3 of SEQ
ID NO: 9; a polynucleotide encoding a polypeptide having 100%
(i.e., 12 out of 12 amino acids) identity to the heavy chain CDR3
of SEQ ID NO: 9; a polynucleotide encoding a polypeptide having at
least 90.9% (i.e., 10 out of 11 amino acids) similarity to the
light chain CDR1 of SEQ ID NO: 4; a polynucleotide encoding a
polypeptide having 100% (i.e., 11 out of 11 amino acids) similarity
to the light chain CDR1 of SEQ ID NO: 4; a polynucleotide encoding
a polypeptide having at least 85.7% (i.e., 6 out of 7 amino acids)
similarity to the light chain CDR2 of SEQ ID NO: 5; a
polynucleotide encoding a polypeptide having 100% (i.e., 7 out of 7
amino acids) similarity to the light chain CDR2 of SEQ ID NO: 5; a
polynucleotide encoding a polypeptide having at least 66.6% (i.e.,
8 out of 12 amino acids) similarity to the light chain CDR3 of SEQ
ID NO: 6; a polynucleotide encoding a polypeptide having at least
75% (i.e., 9 out of 12 amino acids) similarity to the light chain
CDR3 of SEQ ID NO: 6; a polynucleotide encoding a polypeptide
having at least 83.3% (i.e., 10 out of 12 amino acids) similarity
to the light chain CDR3 of SEQ ID NO: 6; a polynucleotide encoding
a polypeptide having at least 91.6% (i.e., 11 out of 12 amino
acids) similarity to the light chain CDR3 of SEQ ID NO: 6; a
polynucleotide encoding a polypeptide having 100% (i.e., 12 out of
12 amino acids) similarity to the light chain CDR3 of SEQ ID NO: 6;
a polynucleotide encoding a polypeptide having at least 80% (i.e.,
4 out of 5 amino acids) similarity to the heavy chain CDR1 of SEQ
ID NO: 7; a polynucleotide encoding a polypeptide having 100%
(i.e., 5 out of 5 amino acids) similarity to the heavy chain CDR1
of SEQ ID NO: 7; a polynucleotide encoding a polypeptide having at
least 56.2% (i.e., 9 out of 16 amino acids) similarity to the heavy
chain CDR2 of SEQ ID NO: 120; a polynucleotide encoding a
polypeptide having at least 62.5% (i.e., 10 out of 16 amino acids)
similarity to the heavy chain CDR2 of SEQ ID NO: 120; a
polynucleotide encoding a polypeptide having at least 68.7% (i.e.,
11 out of 16 amino acids) similarity to the heavy chain CDR2 of SEQ
ID NO: 120; a polynucleotide encoding a polypeptide having at least
75% (i.e., 12 out of 16 amino acids) similarity to the heavy chain
CDR2 of SEQ ID NO: 120; a polynucleotide encoding a polypeptide
having at least 81.2% (i.e., 13 out of 16 amino acids) similarity
to the heavy chain CDR2 of SEQ ID NO: 120; a polynucleotide
encoding a polypeptide having at least 87.5% (i.e., 14 out of 16
amino acids) similarity to the heavy chain CDR2 of SEQ ID NO: 120;
a polynucleotide encoding a polypeptide having at least 93.7%
(i.e., 15 out of 16 amino acids) similarity to the heavy chain CDR2
of SEQ ID NO: 120;
[0289] a polynucleotide encoding a polypeptide having 100% (i.e.,
16 out of 16 amino acids) similarity to the heavy chain CDR2 of SEQ
ID NO: 120; a polynucleotide encoding a polypeptide having at least
50% (i.e., 6 out of 12 amino acids) similarity to the heavy chain
CDR3 of SEQ ID NO: 9; a polynucleotide encoding a polypeptide
having at least 58.3% (i.e., 7 out of 12 amino acids) similarity to
the heavy chain CDR3 of SEQ ID NO: 9; a polynucleotide encoding a
polypeptide having at least 66.6% (i.e., 8 out of 12 amino acids)
similarity to the heavy chain CDR3 of SEQ ID NO: 9; a
polynucleotide encoding a polypeptide having at least 75% (i.e., 9
out of 12 amino acids) similarity to the heavy chain CDR3 of SEQ ID
NO: 9; a polynucleotide encoding a polypeptide having at least
83.3% (i.e., 10 out of 12 amino acids) similarity to the heavy
chain CDR3 of SEQ ID NO: 9; a polynucleotide encoding a polypeptide
having at least 91.6% (i.e., 11 out of 12 amino acids) similarity
to the heavy chain CDR3 of SEQ ID NO: 9; a polynucleotide encoding
a polypeptide having 100% (i.e., 12 out of 12 amino acids)
similarity to the heavy chain CDR3 of SEQ ID NO: 9.
TABLE-US-00001 TABLE 1 Sequences of exemplary anti-IL-6 antibodies.
Antibody chains CDR1 CDR2 CDR3 Antibody PRT. Nuc. PRT. Nuc. PRT.
Nuc. PRT. Nuc. Ab1 light chains * 2 10 4 12 5 13 6 14 20 720 4 12 5
13 6 14 647 721 4 12 5 13 6 14 651 4 12 5 13 6 14 660 662 4 12 5 13
6 14 666 722 4 12 5 13 6 14 699 698 4 694 5 13 6 695 702 701 4 694
5 13 6 695 706 705 4 694 5 13 6 695 709 723 4 12 5 13 6 14 Human
light 648 710 713 chains used in 649 711 714 Ab1 humanization 650
712 715 Ab1 heavy chains 3 11 7 15 8 16 9 17 18 7 15 8 16 9 17 19
724 7 15 120 696 9 17 652 725 7 15 8 16 9 17 656 7 15 8 16 9 17 657
700 7 15 659 696 9 697 658 7 15 120 696 9 17 661 663 7 15 8 16 9 17
664 7 15 8 16 9 17 665 7 15 120 696 9 17 704 703 7 15 120 696 9 697
708 707 7 15 120 696 9 697 Human heavy 653 716 717 chains used in
654 716 717 Ab1 humanization 655 74 82 718 Ab2 light chains 21 29
23 31 24 32 25 33 667 669 23 31 24 32 25 33 Ab2 heavy chains 22 30
26 34 27 35 28 36 668 670 26 34 27 35 28 36 Ab3 light chains 37 45
39 47 40 48 41 49 671 673 39 47 40 48 41 49 Ab3 heavy chains 38 46
42 50 43 51 44 52 672 674 42 50 43 51 44 52 Ab4 light chains 53 61
55 63 56 64 57 65 675 677 55 63 56 64 57 65 Ab4 heavy chains 54 62
58 66 59 67 60 68 676 678 58 66 59 67 60 68 Ab5 light chains 69 77
71 79 72 80 73 81 679 681 71 79 72 80 73 81 Ab5 heavy chains 70 78
74 82 75 83 76 84 680 682 74 82 75 83 76 84 Ab6 light chains 85 93
87 95 88 96 89 97 683 685 87 95 88 96 89 97 Ab6 heavy chains 86 94
90 98 91 99 92 100 684 686 90 98 91 99 92 100 Ab7 light chains 101
109 103 111 104 112 105 113 119 103 111 104 112 105 113 687 689 103
111 104 112 105 113 693 103 111 104 112 105 113 Ab7 heavy chains
102 110 106 114 107 115 108 116 117 106 114 107 115 108 116 118 106
114 121 108 116 688 690 106 114 107 115 108 116 691 106 114 107 115
108 116 692 106 114 121 108 116 Ab8 light chain 122 130 124 132 125
133 126 134 Ab8 heavy chain 123 131 127 135 128 136 129 137 Ab9
light chain 138 146 140 148 141 149 142 150 Ab9 heavy chain 139 147
143 151 144 152 145 153 Ab10 light chain 154 162 156 164 157 165
158 166 Ab10 heavy chain 155 163 159 167 160 168 161 169 Ab11 light
chain 170 178 172 180 173 181 174 182 Ab11 heavy chain 171 179 175
183 176 184 177 185 Ab12 light chain 186 194 188 196 189 197 190
198 Ab12 heavy chain 187 195 191 199 192 200 193 201 Ab13 light
chain 202 210 204 212 205 213 206 214 Ab13 heavy chain 203 211 207
215 208 216 209 217 Ab14 light chain 218 226 220 228 221 229 222
230 Ab14 heavy chain 219 227 223 231 224 232 225 233 Ab15 light
chain 234 242 236 244 237 245 238 246 Ab15 heavy chain 235 243 239
247 240 248 241 249 Ab16 light chain 250 258 252 260 253 261 254
262 Ab16 heavy chain 251 259 255 263 256 264 257 265 Ab17 light
chain 266 274 268 276 269 277 270 278 Ab17 heavy chain 267 275 271
279 272 280 273 281 Ab18 light chain 282 290 284 292 285 293 286
294 Ab18 heavy chain 283 291 287 295 288 296 289 297 Ab19 light
chain 298 306 300 308 301 309 302 310 Ab19 heavy chain 299 307 303
311 304 312 305 313 Ab20 light chain 314 322 316 324 317 325 318
326 Ab20 heavy chain 315 323 319 327 320 328 321 329 Ab21 light
chain 330 338 332 340 333 341 334 342 Ab21 heavy chain 331 339 335
343 336 344 337 345 Ab22 light chain 346 354 348 356 349 357 350
358 Ab22 heavy chain 347 355 351 359 352 360 353 361 Ab23 light
chain 362 370 364 372 365 373 366 374 Ab23 heavy chain 363 371 367
375 368 376 369 377 Ab24 light chain 378 386 380 388 381 389 382
390 Ab24 heavy chain 379 387 383 391 384 392 385 393 Ab25 light
chain 394 402 396 404 397 405 398 406 Ab25 heavy chain 395 403 399
407 400 408 401 409 Ab26 light chain 410 418 412 420 413 421 414
422 Ab26 heavy chain 411 419 415 423 416 424 417 425 Ab27 light
chain 426 434 428 436 429 437 430 438 Ab27 heavy chain 427 435 431
439 432 440 433 441 Ab28 light chain 442 450 444 452 445 453 446
454 Ab28 heavy chain 443 451 447 455 448 456 449 457 Ab29 light
chain 458 466 460 468 461 469 462 470 Ab29 heavy chain 459 467 463
471 464 472 465 473 Ab30 light chain 474 482 476 484 477 485 478
486 Ab30 heavy chain 475 483 479 487 480 488 481 489 Ab31 light
chain 490 498 492 500 493 501 494 502 Ab31 heavy chain 491 499 495
503 496 504 497 505 Ab32 light chain 506 514 508 516 509 517 510
518 Ab32 heavy chain 507 515 511 519 512 520 513 521 Ab33 light
chain 522 530 524 532 525 533 526 534 Ab33 heavy chain 523 531 527
535 528 536 529 537 Ab34 light chain 538 546 540 548 541 549 542
550 Ab34 heavy chain 539 547 543 551 544 552 545 553 Ab35 light
chain 554 562 556 564 557 565 558 566 Ab35 heavy chain 555 563 559
567 560 568 561 569 Ab36 light chain 570 578 572 580 573 581 574
582 Ab36 heavy chain 571 579 575 583 576 584 577 585 * Exemplary
sequence variant forms of heavy and light chains are shown on
separate lines. PRT.: Polypeptide sequence. Nuc.: Exemplary coding
sequence.
[0290] For reference, sequence identifiers other than those
included in Table 1 are summarized in Table 2.
TABLE-US-00002 TABLE 2 Summary of sequence identifiers in this
application. SEQ ID Description 1 Human IL-6 586 kappa constant
light chain polypeptide sequence 587 kappa constant light chain
polynucleotide sequence 588 gamma-1 constant heavy chain
polypeptide sequence 589 gamma-1 constant heavy chain
polynucleotide sequence 590-646 Human IL-6 peptides (Example 14)
719 gamma-1 constant heavy chain polypeptide sequence (differs from
SEQ ID NO: 518 at two positions) 726 C-reactive protein polypeptide
sequence 727 IL-6 receptor alpha 728 IL-6 receptor beta/gp130
[0291] Such antibody fragments or variants thereof may be present
in at least one of the following non-limiting forms: Fab, Fab',
F(ab').sub.2, Fv and single chain Fv antibody forms. In a preferred
embodiment, the anti-IL-6 antibodies described herein further
comprises the kappa constant light chain sequence comprising the
sequence set forth in the polypeptide sequence of SEQ ID NO:
586.
[0292] In another preferred embodiment, the anti-IL-6 antibodies
described herein further comprises the gamma-1 constant heavy chain
polypeptide sequence comprising one of the sequences set forth in
the polypeptide sequence of SEQ ID NO: 588 and SEQ ID NO: 719.
[0293] Embodiments of antibodies described herein may include a
leader sequence, such as a rabbit Ig leader, albumin pre-peptide, a
yeast mating factor pre pro secretion leader sequence (such as P.
pastoris or Saccharomyces cerevisiae a or alpha factor), or human
HAS leader. Exemplary leader sequences are shown offset from FR1 at
the N-terminus of polypeptides shown in FIGS. 10A-B and 11A-B as
follows: rabbit Ig leader sequences in SEQ ID NOs: 2 and 660 and
SEQ ID NOs: 3 and 661; and an albumin prepeptide in SEQ ID NOs: 706
and 708, which facilitates secretion. Other leader sequences known
in the art to confer desired properties, such as secretion,
improved stability or half-life, may also be used, either alone or
in combinations with one another, on the heavy and/or light chains,
which may optionally be cleaved prior to administration to a
subject. For example, a polypeptide may be expressed in a cell or
cell-free expression system that also expresses or includes (or is
modified to express or include) a protease, e.g., a membrane-bound
signal peptidase, that cleaves a leader sequence.
[0294] In another embodiment, the subject technology contemplates
an isolated anti-IL-6 antibody comprising a V.sub.H polypeptide
sequence comprising: SEQ ID NO: 3, 18, 19, 22, 38, 54, 70, 86, 102,
117, 118, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283,
299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491,
507, 523, 539, 555, 571, 652, 656, 657, 658, 661, 664, 665, 668,
672, 676, 680, 684, 688, 691, 692, 704, or 708; and further
comprising a V.sub.L polypeptide sequence comprising: SEQ ID NO: 2,
20, 21, 37, 53, 69, 85, 101, 119, 122, 138, 154, 170, 186, 202,
218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410,
426, 442, 458, 474, 490, 506, 522, 538, 554, 570, 647, 651, 660,
666, 667, 671, 675, 679, 683, 687, 693, 699, 702, 706, or 709 or a
variant thereof wherein at least one of the framework residues (FR
residues) or CDR residues in said V.sub.H or V.sub.L polypeptide
has been substituted with another amino acid residue resulting in
an anti-IL-6 antibody that specifically binds IL-6. The subject
technology contemplates humanized and chimeric forms of these
antibodies wherein preferably the FR will comprise human FRs highly
homologous to the parent antibody. The chimeric antibodies may
include an Fc derived from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6,
IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16,
IgG17, IgG18 or IgG19 constant regions and in particular a variable
heavy and light chain constant region as set forth in SEQ ID NO:
588 and SEQ ID NO: 586.
[0295] In one embodiment of the subject technology, the antibodies
or V.sub.H or V.sub.L polypeptides originate or are selected from
at least one rabbit B cell populations prior to initiation of the
humanization process referenced herein.
[0296] In another embodiment of the subject technology, the
anti-IL-6 antibodies and fragments and variants thereof have
binding specificity for primate homologs of the human IL-6 protein.
Non-limiting examples of primate homologs of the human IL-6 protein
are IL-6 obtained from Macaca fascicularis (cynomolgus monkey) and
the Rhesus monkey. In another embodiment of the subject technology,
the anti-IL-6 antibodies and fragments and variants thereof
inhibits the association of IL-6 with IL-6R, and/or the production
of IL-6/IL-6R/130 complexes and/or the production of
IL-6/IL-6R/gp130 multimers and/or antagonizes the biological
effects of at least one of the foregoing.
Polyclonal Antibody
[0297] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen. Polyclonal antibodies which selectively bind the IL-6
may be made by methods well-known in the art. See, e.g., Howard
& Kaser (2007) Making and Using Antibodies: A Practical
Handbook CRC Press.
Monoclonal Antibody
[0298] A monoclonal antibody contains a substantially homogeneous
population of antibodies specific to antigens, which population
contains substantially similar epitope binding sites. Monoclonal
antibodies may be obtained by methods known to those skilled in the
art. See, e.g. Kohler and Milstein (1975) Nature 256: 495-497; U.S.
Pat. No. 4,376,110; Ausubel, et al. [Eds.] (2011) CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, Greene Publishing Assoc. and Wiley
Interscience, NY.; and Harlow & Lane (1998) USING ANTIBODIES: A
LABORATORY MANUAL Cold Spring Harbor Laboratory; Colligan, et al.
(2005) [Eds.] Current Protocols in Immunology Greene Publishing
Assoc. and Wiley Interscience, NY. Such antibodies may be of any
immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any
subclass thereof. A hybridoma producing an antibody of the present
subject technology may be cultivated in vitro, in situ, or in
vivo.
Chimeric Antibody
[0299] Chimeric antibodies are molecules different portions of
which are derived from different animal species, such as those
having variable region derived from a murine antibody and a human
immunoglobulin constant region, which are primarily used to reduce
immunogenicity in application and to increase yields in production,
for example, where murine monoclonal antibodies have higher yields
from hybridomas but higher immunogenicity in humans, such that
human murine chimeric monoclonal antibodies are used. Chimeric
antibodies and methods for their production are known in the art.
See Cabilly, et al. (1984) Proc. Natl. Acad. Sci. USA 81:
3273-3277; Morrison, et al. (1994) Proc. Natl. Acad. Sci. USA 81:
6851-6855, Boulianne, et al. (1984) Nature 312: 643-646; Neuberger,
et al. (1985) Nature 314: 268-270; European Patent Application
173494 (1986); WO 86/01533 (1986); European Patent Application
184187 (1986); European Patent Application 73494 (1986); Sahagan,
et al. (1986) J Immunol. 137: 1066-1074; Liu, et al. (1987) Proc.
Natl. Acad. Sci. USA 84: 3439-3443; Sun, et al. (1987) Proc. Natl.
Acad. Sci. USA 84: 214-218; Better, et al. (1988) Science 240:
1041-1043; and Harlow & Lane (1998) USING ANTIBODIES: A
LABORATORY MANUAL Cold Spring Harbor Laboratory; and U.S. Pat. No.
5,624,659.
Humanized Antibody
[0300] Humanized antibodies are engineered to contain even more
human-like immunoglobulin domains, and incorporate only the
complementarity-determining regions of the animal-derived antibody.
This may be accomplished by examining the sequence of the
hyper-variable loops of the variable regions of the monoclonal
antibody, and fitting them to the structure of the human antibody
chains. See, e.g., U.S. Pat. No. 6,187,287. Likewise, other methods
of producing humanized antibodies are now well known in the art.
See, e.g., U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089;
5,693,762; 6,054,297; 6,180,370; 6,407,213; 6,548,640; 6,632,927;
and 6,639,055; Jones, et al. (1986) Nature 321: 522-525; Reichmann,
et al. (1988) Nature 332: 323-327; Verhoeyen, et al. (1988) Science
239: 1534-36; and Zhiqiang An (2009) [Ed.] Therapeutic Monoclonal
Antibodies: From Bench to Clinic John Wiley & Sons, Inc.
Antibody Fragments (Antigen-Binding Fragments)
[0301] In addition to entire immunoglobulins (or their recombinant
counterparts), immunoglobulin fragments comprising the epitope
binding site (e.g., Fab', F(ab').sub.2, or other fragments) may be
synthesized. "Fragment," or minimal immunoglobulins may be designed
utilizing recombinant immunoglobulin techniques. For instance "Fv"
immunoglobulins for use in the present subject technology may be
produced by synthesizing a fused variable light chain region and a
variable heavy chain region. Combinations of antibodies are also of
interest, e.g. diabodies, which comprise two distinct Fv
specificities. Antigen-binding fragments of immunoglobulins include
but are not limited to SMIPs (small molecule
immunopharmaceuticals), camelbodies, nanobodies, and IgNAR.
Anti-Idiotypic Antibody
[0302] An anti-idiotypic (anti-Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an antibody. An Id antibody may be prepared
by immunizing an animal of the same species and genetic type (e.g.,
mouse strain) as the source of the antibody with the antibody to
which an anti-Id is being prepared. The immunized animal will
recognize and respond to the idiotypic determinants of the
immunizing antibody by producing an antibody to these idiotypic
determinants (the anti-ld antibody). See e.g., U.S. Pat. No.
4,699,880. The anti-Id antibody may also be used as an "immunogen"
to induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody. The anti-anti-Id may be
epitopically identical to the original antibody which induced the
anti-Id. Thus, by using antibodies to the idiotypic determinants of
an antibody it is possible to identify other clones expressing
antibodies of identical specificity.
Engineered and Modified Antibodies
[0303] An antibody of the subject technology further may be
prepared using an antibody having at least one of the V.sub.H
and/or V.sub.L sequences derived from an antibody starting material
to engineer a modified antibody, which modified antibody may have
altered properties from the starting antibody. An antibody may be
engineered by modifying at least one residues within one or both
variable regions (i.e., V.sub.H and/or V.sub.L), for example within
at least one CDR regions and/or within at least one framework
regions. Additionally or alternatively, an antibody may be
engineered by modifying residues within the constant region(s), for
example to alter the effector function(s) of the antibody.
[0304] One type of variable region engineering that may be
performed is CDR grafting. Antibodies interact with target antigens
predominantly through amino acid residues that are located in the
six heavy and light chain complementarity determining regions
(CDRs). For this reason, the amino acid sequences within CDRs are
more diverse between individual antibodies than sequences outside
of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties. See, e.g., Riechmann, et al. (1998)
Nature 332: 323-327; Jones, et al. (1986) Nature 321: 522-525;
Queen, et al. (1989) Proc. Natl. Acad. U.S.A. 86: 10029-10033; U.S.
Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762; and
6,180,370.
[0305] Suitable framework sequences may be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes may be found in the "VBase"
human germline sequence database (available on the Internet), as
well as in Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242; Tomlinson, et al.
(1992) "The Repertoire of Human Germline V.sub.H Sequences Reveals
about Fifty Groups of V.sub.H Segments with Different Hypervariable
Loops" J. Mol. Biol. 227: 776-798; and Cox, et al. (1994) Eur. J
Immunol. 24: 827-836.
[0306] Another type of variable region modification is to mutate
amino acid residues within the V.sub.H and/or V.sub.L CDR 1, CDR2
and/or CDR3 regions to thereby improve at least one binding
properties (e.g., affinity) of the antibody of interest.
Site-directed mutagenesis or PCR-mediated mutagenesis may be
performed to introduce the mutation(s) and the effect on antibody
binding, or other functional property of interest, may be evaluated
in appropriate in vitro or in vivo assays. Preferably conservative
modifications (as discussed herein) may be introduced. The
mutations may be amino acid substitutions, additions or deletions,
but are preferably substitutions. Moreover, typically no more than
one, two, three, four or five residues within a CDR region are
altered.
[0307] Engineered antibodies of the subject technology include
those in which modifications have been made to framework residues
within V.sub.H and/or V.sub.L, e.g. to improve the properties of
the antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "backmutate" at least one framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues may be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived.
[0308] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the subject technology may
be engineered to include modifications within the Fc region,
typically to alter at least one functional properties of the
antibody, such as serum half-life, complement fixation, Fc receptor
binding, and/or antigen-dependent cellular cytotoxicity.
Furthermore, an antibody of the subject technology may be
chemically modified (e.g., at least one chemical moieties may be
attached to the antibody) or be modified to alter its
glycosylation, again to alter at least one functional properties of
the antibody. Such embodiments are described further below. The
numbering of residues in the Fc region is that of the EU index of
Kabat.
[0309] The hinge region of CH1 may be modified such that the number
of cysteine residues in the hinge region is altered, e.g.,
increased or decreased. See U.S. Pat. No. 5,677,425. The number of
cysteine residues in the hinge region of CH1 may be altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the antibody. The Fc hinge
region of an antibody may be mutated to decrease the biological
half-life of the antibody. More specifically, at least one amino
acid mutations may be introduced into the CH2-CH3 domain interface
region of the Fc-hinge fragment such that the antibody has impaired
Staphylococcyl protein A (SpA) binding relative to native Fc-hinge
domain SpA binding. See, e.g., U.S. Pat. No. 6,165,745.
[0310] The antibody may be modified to increase its biological
half-life. Various approaches are possible. For example, at least
one of the following mutations may be introduced: T252L, T254S,
T256F. See U.S. Pat. No. 6,277,375. Alternatively, to increase the
biological half-life, the antibody may be altered within the CH1 or
CL region to contain a salvage receptor binding epitope taken from
two loops of a CH2 domain of an Fc region of an IgG. See U.S. Pat.
Nos. 5,869,046 and 6,121,022.
[0311] The Fc region may be altered by replacing at least one amino
acid residue with a different amino acid residue to alter the
effector function(s) of the antibody. For example, at least one
amino acid selected from amino acid residues 234, 235, 236, 237,
297, 318, 320 and 322 may be replaced with a different amino acid
residue such that the antibody has an altered affinity for an
effector ligand but retains the antigen-binding ability of the
parent antibody. The effector ligand to which affinity may be
altered may be, for example, an Fc receptor or the Cl component of
complement. See U.S. Pat. Nos. 5,624,821 and 5,648,260.
[0312] The Fc region may be modified to increase the affinity of
the antibody for an Fcy receptor by modifying at least one amino
acids at the following positions: 238, 239, 248, 249, 252, 254,
255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283,
285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305,
307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,
334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398,
414, 416, 419, 430, 434, 435, 437, 438 or 439. See WO 00/42072.
Moreover, the binding sites on human IgG1 for Fc.gamma.RI,
Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped and variants
with improved binding. See Shields, et al. (2001) J. Biol. Chem.
276: 6591-6604. Specific mutations at positions 256, 290, 298, 333,
334 and 339 are shown to improve binding to Fc.gamma.RIII.
Additionally, the following combination mutants are shown to
improve Fc.gamma.RIII binding: T256A/S298A, S298A/E333A,
S298A/K224A and S298A/E333A/K334A.
[0313] The glycosylation of an antibody may be modified. For
example, an aglycosylated antibody may be made (i.e., the antibody
lacks glycosylation). Glycosylation may be altered to, for example,
increase the affinity of the antibody for antigen. Such
carbohydrate modifications may be accomplished by, for example,
altering at least one sites of glycosylation within the antibody
sequence. For example, at least one amino acid substitutions may be
made that result in elimination of at least one variable region
framework glycosylation sites to thereby eliminate glycosylation at
that site. Such aglyclosylation may increase the affinity of the
antibody for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and
6,350,861.
[0314] Additionally or alternatively, an antibody may be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such carbohydrate
modifications may be accomplished by, for example, expressing the
antibody in a host cell with altered glycosylation machinery. Cells
with altered glycosylation machinery have been described in the art
and may be used as host cells in which to express recombinant
antibodies of the subject technology to thereby produce an antibody
with altered glycosylation. See U.S. Patent Application Publication
No. 2004/0110704 and Yamane-Ohnuki, et al. (2004) Biotechnol
Bioeng. 87: 614-22; EP 1,176,195; WO 2003/035835; Shields, et al.
(2002) J. Biol. Chem. 277: 26733-26740; WO 99/54342; Umana, et al.
(1999) Nat. Biotech. 17: 176-180; and Tarentino, et al. (1975)
Biochem. 14: 5516-23.
[0315] An antibody may be pegylated to, for example, increase the
biological (e.g., serum) half-life of the antibody. To pegylate an
antibody, the antibody, or fragment thereof, typically is reacted
with polyethylene glycol (PEG), such as a reactive ester or
aldehyde derivative of PEG, under conditions in which at least one
PEG groups become attached to the antibody or antibody fragment.
Preferably, the pegylation is carried out via an acylation reaction
or an alkylation reaction with a reactive PEG molecule (or an
analogous reactive water-soluble polymer).
[0316] The subject technology also provides variants and
equivalents that are substantially homologous to the antibodies,
antibody fragments, diabodies, SMIPs, camelbodies, nanobodies,
IgNAR, polypeptides, variable regions and CDRs set forth herein.
These may contain, e.g., conservative substitution mutations,
(i.e., the substitution of at least one amino acids by similar
amino acids). For example, conservative substitution refers to the
substitution of an amino acid with another within the same general
class, e.g., one acidic amino acid with another acidic amino acid,
one basic amino acid with another basic amino acid, or one neutral
amino acid by another neutral amino acid. In another embodiment,
the subject technology further contemplates the above-recited
polypeptide homologs of the antibody fragments, variable regions
and CDRs set forth herein further having anti-IL-6 activity.
Non-limiting examples of anti-IL-6 activity are set forth herein,
for example, under the heading "Anti-IL-6 Activity," infra.
[0317] Anti-IL-6 antibodies have also been disclosed in the
following published and unpublished patent applications, which are
co-owned by the assignee of the present application: WO
2008/144763; U.S. Patent Application Publication Nos. 2009/0028784,
2009/0297513, and 2009/0297436. Other anti-IL-6 antibodies have
been disclosed in the following U.S. Patents and Published Patent
Application Nos: U.S. Pat. Nos. 7,482,436; 7,291,721; 6,121,423;
2008/0075726; 2007/0178098; 2007/0154481; 2006/0257407; and
2006/0188502.
Polypeptide Sequence Variants
[0318] For any anti-IL-6 antibodies sequence described herein,
further characterization or optimization may be achieved by
systematically either adding or removing amino acid residues to
generate longer or shorter peptides, and testing those and
sequences generated by walking a window of the longer or shorter
size up or down the antigen from that point. Coupling this approach
to generating new candidate targets with testing for effectiveness
of antigenic molecules based on those sequences in an
immunogenicity assay, as known in the art or as described herein,
may lead to further manipulation of the antigen. Further still,
such optimized sequences may be adjusted by, e.g., the addition,
deletions, or other mutations as known in the art and/or discussed
herein to further optimize the anti-IL-6 antibodies (e.g.,
increasing serum stability or circulating half-life, increasing
thermal stability, enhancing delivery, enhance immunogenicity,
increasing solubility, targeting to a particular in vivo location
or cell type).
[0319] In another embodiment, the subject technology contemplates
polypeptide sequences having at least about 90% sequence homology
to any at least one of the polypeptide sequences of antibody
fragments, variable regions and CDRs set forth herein. More
preferably, the subject technology contemplates polypeptide
sequences having at least about 95% sequence homology, even more
preferably at least about 98% sequence homology, and still more
preferably at least about 99% sequence homology to any at least one
of the polypeptide sequences of antibody fragments, variable
regions and CDRs set forth herein. Methods for determining homology
between nucleic acid and amino acid sequences are well known to
those of ordinary skill in the art.
[0320] The anti-IL-6 antibodies polypeptides described herein may
comprise conservative substitution mutations, (i.e., the
substitution of at least one amino acids by similar amino acids).
For example, conservative substitution refers to the substitution
of an amino acid with another within the same general class, e.g.,
one acidic amino acid with another acidic amino acid, one basic
amino acid with another basic amino acid, or one neutral amino acid
by another neutral amino acid.
[0321] Anti-IL-6 antibodies polypeptide sequences may have at least
about 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 98.5, 99, 99.5, 99.8, 99.9, or 100%
sequence homology to any at least one of the polypeptide sequences
set forth herein. More preferably, the subject technology
contemplates polypeptide sequences having at least about 95%
sequence homology, even more preferably at least about 98% sequence
homology, and still more preferably at least about 99% sequence
homology to any at least one of the polypeptide sequences of
Anti-IL-6 antibodies polypeptide sequences set forth herein.
Methods for determining homology between amino acid sequences, as
well as nucleic acid sequences, are well known to those of ordinary
skill in the art. See, e.g., Nedelkov & Nelson (2006) New and
Emerging Proteomic Techniques Humana Press. Thus, an anti-IL-6
antibodies polypeptide may have at least about 60, 65, 70, 75, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 98.5, 99, 99.5, 99.8, 99.9, or 100% sequence homology with a
polypeptide sequence.
[0322] The term homology, or identity, is understood as meaning the
number of agreeing amino acids (identity) with other proteins,
expressed in percent. The identity is preferably determined by
comparing a given sequence with other proteins with the aid of
computer programs. If sequences which are compared with each other
are different in length, the identity is to be determined in such a
way that the number of amino acids which the short sequence shares
with the longer sequence determines the percentage identity. The
identity can be determined routinely by means of known computer
programs which are publicly available such as, for example,
ClustalW. Thompson, et al. (1994) Nucleic Acids Research 22:
4673-4680. ClustalW is publicly available from the European
Molecular Biology Laboratory and may be downloaded from various
internet pages, inter alia the IGBMC (Institut de Genetique et de
Biologie Moleculaire et Cellulaire) and the EBI and all mirrored
EBI internet pages (European Bioinformatics Institute). If the
ClustalW computer program Version 1.8 is used to determine the
identity between, for example, the reference protein of the present
application and other proteins, the following parameters are to be
set: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10,
GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF),
NOPGAP, NOHGAP. See also European Bioinformatics Institute (EBI)
toolbox available on-line and Smith (2002) Protein Sequencing
Protocols [2.sup.11'' Ed.] Humana Press.
[0323] One possibility of finding similar sequences is to carry out
sequence database researches. Here, at least one sequences may be
entered as what is known as a query. This query sequence is then
compared with sequences present in the selected databases using
statistical computer programs. Such database queries (blast
searches) are known to the skilled worker and may be carried out at
different suppliers. If, for example, such a database query is
carried out at the NCBI (National Center for Biotechnology
Information), the standard settings for the respective comparison
query should be used. For protein sequence comparisons (blastp),
these settings are: Limit entrez=not activated; Filter=low
complexity activated; Expect value=10; word size=3;
Matrix=BLOSUM62; Gap costs: Existence=11, Extension=1. The result
of such a query is, among other parameters, the degree of identity
between the query sequence and the similar sequences found in the
databases. Methods and materials for making fragments of Anti-IL-6
antibodies polypeptides are well known in the art. See, e.g.,
Maniatis, et al. (2001) Molecular Cloning: A Laboratory
Manual[3.sup.rd Ed.] Cold Spring Harbor Laboratory Press.
[0324] Variant anti-IL-6 antibodies polypeptides may retain their
antigenic specificity to bind IL-6. Fully specific variants may
contain only conservative variations or variations in non-critical
residues or in non-critical regions. Variants may also contain
substitution of similar amino acids that result in no change or an
insignificant change in their specificity. Alternatively, such
substitutions may positively or negatively affect specificity to
some degree. Non-specific variants typically contain at least one
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region of an epitope.
Molecular biology and biochemistry techniques for modifying
anti-IL-6 antibodies polypeptides while preserving specificity are
well known in the art. See, e.g., Ho, et al. (1989) Gene 77(1):
51-59; Landt, et al. (1990) Gene 96(1): 125-128; Hopp & Woods
(1991) Proc. Natl. Acad. Sci. USA 78(6): 3824-3828; Kolaskar &
Tongaonkar (1990) FEBS Letters 276(1-2): 172-174; and Welling, et
al. (1985) FEBS Letters 188(2): 215-218
[0325] Amino acids that are essential for function may be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis. Cunningham, et al.
(1989) Sci. 244: 1081-85. The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity such as
epitope binding. Sites that are critical for ligand-receptor
binding may also be determined by structural analysis such as
crystallography, nuclear magnetic resonance, or photoaffinity
labeling. Smith, et al. (1992) J. Mol. Biol. 224: 899-904; de Vos,
et al. (1992) Sci. 255: 306-12.
[0326] For example, one class of substitutions is conserved amino
acid substitutions. Such substitutions are those that substitute a
given amino acid in an anti-IL-6 antibody polypeptide with another
amino acid of like characteristics. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr, exchange of the acidic residues Asp
and Glu, substitution between the amide residues Asn and Gln,
exchange of the basic residues Lys and Arg, replacements among the
aromatic residues Phe, Tyr. Guidance concerning which amino acid
changes are likely to be phenotypically silent is found in, for
example, Bowie, et al. (1990) Sci. 247: 1306-10. Hence, one of
ordinary skill in the art appreciates that the inventors possess
peptide variants without delineation of all the specific variants.
As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the subject technology. See, e.g., Creighton (1992)
Proteins: Structures and Molecular Properties [2.sup.nd Ed.] W.H.
Freeman.
[0327] Moreover, polypeptides often contain amino acids other than
the twenty "naturally occurring" amino acids. Further, many amino
acids, including the terminal amino acids, may be modified by
natural processes, such as processing and other post-translational
modifications, or by chemical modification techniques well known in
the art. Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, g-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination. See
Creighton (1992) Proteins: Structure and Molecular Properties
[2.sup.nd Ed.] and Lundblad (1995) Techniques in Protein
Modification[1.sup.st Ed.] Many detailed reviews are available on
this subject. See, e.g., Wold (1983) Posttranslational Covalent
Modification of Proteins Acad. Press, NY; Seifter, et al. (1990)
Meth. Enzymol. 182: 626-46; and Rattan, et al. (1992) Ann. NY Acad.
Sci. 663: 48-62.
[0328] In another embodiment, the subject technology further
contemplates the generation and use of anti-idiotypic antibodies
that bind any of the foregoing sequences. In an exemplary
embodiment, such an anti-idiotypic antibody could be administered
to a subject who has received an anti-IL-6 antibody to modulate,
reduce, or neutralize, the effect of the anti-IL-6 antibody. A
further exemplary use of such anti-idiotypic antibodies is for
detection of the anti-IL-6 antibodies of the present subject
technology, for example to monitor the levels of the anti-IL-6
antibodies present in a subject's blood or other bodily fluids.
[0329] The present subject technology also contemplates anti-IL-6
antibodies comprising any of the polypeptide or polynucleotide
sequences described herein substituted for any of the other
polynucleotide sequences described herein. For example, without
limitation thereto, the present subject technology contemplates
antibodies comprising the combination of any of the variable light
chain and variable heavy chain sequences described herein, and
further contemplates antibodies resulting from substitution of any
of the CDR sequences described herein for any of the other CDR
sequences described herein. As noted preferred anti-IL-6 antibodies
or fragments or variants thereof may contain a variable heavy
and/or light sequence as shown in FIG. 2-5, such as SEQ ID NO:
651,657,709 or variants thereof wherein at least one CDR or FR
residues are modified without adversely affecting antibody binding
to IL-6 or other desired functional activity.
Fusion Proteins
[0330] Fusions comprising the anti-IL-6 antibodies polypeptides are
also within the scope of the present subject technology. For
example, the fusion protein may be linked to a GST fusion protein
in which the anti-IL-6 antibodies polypeptide sequences are fused
to the C-terminus of the GST sequences. Such fusion proteins may
facilitate the purification of the recombinant Anti-IL-6 antibodies
polypeptides. Alternatively, anti-IL-6 antibodies polypeptides may
be fused with a protein that binds B-cell follicles, thus
initiating both a humoral immune response and activation of T
cells. Berney, et al. (1999) J. Exp. Med. 190: 851-60.
Alternatively, for example, the Anti-IL-6 antibodies polypeptides
may be genetically coupled with and anti-dendritic cell antibody to
deliver the antigen to the immune system and stimulate a cellular
immune response. He, et al. (2004) Clin. Cancer Res. 10: 1920-27. A
chimeric or fusion protein of the subject technology may be
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are
ligated together in-frame in accordance with conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini
for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. The fusion gene may be synthesized by
conventional techniques including automated DNA synthesizers.
[0331] Fusion proteins may include C-terminal or N-terminal
translocation sequences. Further, fusion proteins can comprise
additional elements, e.g., for protein detection, purification, or
other applications. Detection and purification facilitating domains
including but not limited to metal chelating peptides such as
polyhistidine tracts, histidine-tryptophan modules, or other
domains that allow purification on immobilized metals; maltose
binding protein; protein A domains that allow purification on
immobilized immunoglobulin; or the domain utilized in the FLAG
extension/affinity purification system (Immunex Corp, Seattle
Wash.)
[0332] A fusion protein may be prepared from a protein of the
subject technology by fusion with a portion of an immunoglobulin
comprising a constant region of an immunoglobulin. More preferably,
the portion of the immunoglobulin comprises a heavy chain constant
region which is optionally and more preferably a human heavy chain
constant region. The heavy chain constant region is most preferably
an IgG heavy chain constant region, and optionally and most
preferably is an Fc chain, most preferably an IgG Fc fragment that
comprises CH2 and CH3 domains. Although any IgG subtype may
optionally be used, the IgG1 subtype is preferred. The Fc chain may
optionally be a known or "wild type" Fc chain, or alternatively may
be mutated. See, e.g., U.S. Patent Application Publication No.
2006/0034852. The term "Fc chain" also optionally comprises any
type of Fc fragment. Several of the specific amino acid residues
that are involved in antibody constant region-mediated activity in
the IgG subclass have been identified. Inclusion, substitution or
exclusion of these specific amino acids therefore allows for
inclusion or exclusion of specific immunoglobulin constant
region-mediated activity. Furthermore, specific changes may result
in aglycosylation for example and/or other desired changes to the
Fc chain. At least some changes may optionally be made to block a
function of Fc which is considered to be undesirable, such as an
undesirable immune system effect. See McCafferty, et al. (2002)
Antibody Engineering: A Practical Approach (Eds.) Oxford University
Press.
[0333] The inclusion of a cleavable linker sequences such as Factor
Xa (see, e.g., Ottavi, (1998) Biochimie 80: 289-93), subtilisin
protease recognition motif (see, e.g., Polyak (1997) Protein Eng.
10: 615-19); enterokinase (Invitrogen, San Diego, Calif.), between
the translocation domain (for efficient plasma membrane expression)
and the rest of the newly translated polypeptide may be useful to
facilitate purification. For example, one construct can include a
polypeptide encoding a nucleic acid sequence linked to six
histidine residues followed by a thioredoxin, an enterokinase
cleavage site (see, e.g., Williams (1995) Biochemistry 34:
1787-97), and an C-terminal translocation domain. The histidine
residues facilitate detection and purification while the
enterokinase cleavage site provides a means for purifying the
desired protein(s) from the remainder of the fusion protein.
Technology pertaining to vectors encoding fusion proteins and
application of fusion proteins are well described in the scientific
and patent literature. See, e.g., Kroll (1993) DNA Cell. Biol. 12:
441-53.
Conjugates
[0334] The anti-IL-6 antibodies, antibodies that bind the Anti-IL-6
antibodies and fragments thereof, may be conjugated to other
moieties. Such conjugates are often used in the preparation of
vaccines. The anti-IL-6 antibodies polypeptide may be conjugated to
a carbohydrate (e.g., mannose, fucose, glucose, GlcNAs, maltose),
which is recognized by the mannose receptor present on dendritic
cells and macrophages. The ensuing binding, aggregation, and
receptor-mediated endocytosis and phagocytosis functions provide
enhanced innate and adaptive immunity. See Mahnke, et al. (2000) J.
Cell Biol. 151: 673-84; Dong, et al. (1999) J. Immunol. 163:
5427-34. Other moieties suitable for conjugation to elicit an
immune response includes but not limited to Keyhole Limpet
Hemocyanin (KLH), diphtheria toxoid, cholera toxoid, Pseudomonas
exoprotein A, and microbial outer membrane proteins (OMPS).
Polynucleotides Encoding Anti-IL-6 Antibody Polypeptides
[0335] The subject technology is further directed to
polynucleotides encoding polypeptides of the antibodies having
binding specificity to IL-6. In one embodiment of the subject
technology, polynucleotides of the subject technology comprise, or
alternatively consist of, the following polynucleotide sequence
encoding the variable light chain polypeptide sequence of SEQ ID
NO: 2 which is encoded by the polynucleotide sequence of SEQ ID NO:
10 or the polynucleotide sequence of SEQ ID NO: 662,698,701, or
705.
[0336] In another embodiment of the subject technology,
polynucleotides of the subject technology comprise, or
alternatively consist of, the following polynucleotide sequence
encoding the variable heavy chain polypeptide sequence of SEQ ID
NO: 3 which is encoded by the polynucleotide sequence of SEQ ID NO:
11 or the polynucleotide sequence of SEQ ID NO: 663, 700, 703, or
707.
[0337] In a further embodiment of the subject technology,
polynucleotides encoding fragments or variants of the antibody
having binding specificity to IL-6 comprise, or alternatively
consist of, at least one of the polynucleotide sequences of SEQ ID
NO: 12 or 694; SEQ ID NO: 13; and SEQ ID NO: 14 or 695 which
correspond to polynucleotides encoding the
complementarity-determining regions (CDRs, or hypervariable
regions) of the light chain variable sequence of SEQ ID NO: 2.
[0338] In a further embodiment of the subject technology,
polynucleotides encoding fragments or variants of the antibody
having binding specificity to IL-6 comprise, or alternatively
consist of, at least one of the polynucleotide sequences of SEQ ID
NO: 15; SEQ ID NO: 16 or 696; and SEQ ID NO: 17 or 697 which
correspond to polynucleotides encoding the
complementarity-determining regions (CDRs, or hypervariable
regions) of the heavy chain variable sequence of SEQ ID NO: 3 or
SEQ ID NO: 661 or SEQ ID NO: 657 or others depicted in FIG. 8 or
9.
[0339] The subject technology also contemplates polynucleotide
sequences including at least one of the polynucleotide sequences
encoding antibody fragments or variants described herein. In one
embodiment of the subject technology, polynucleotides encoding
fragments or variants of the antibody having binding specificity to
IL-6 comprise, or alternatively consist of, one, two, three or
more, including all of the following polynucleotides encoding
antibody fragments: the polynucleotide SEQ ID NO: 10 encoding the
light chain variable region of SEQ ID NO: 2; the polynucleotide SEQ
ID NO: 11 encoding the heavy chain variable region of SEQ ID NO: 3;
the polynucleotide SEQ ID NO: 720 encoding the light chain
polypeptide of SEQ ID NO: 20; the polynucleotide SEQ ID NO: 721
encoding the light chain polypeptide of SEQ ID NO: 647; the
polynucleotide SEQ ID NO: 662 encoding the light chain polypeptide
of SEQ ID NO: 660; the polynucleotide SEQ ID NO: 722 encoding the
light chain polypeptide of SEQ ID NO: 666; the polynucleotide SEQ
ID NO: 698 encoding the light chain polypeptide of SEQ ID NO: 699;
the polynucleotide SEQ ID NO: 701 encoding the light chain
polypeptide of SEQ ID NO: 702; the polynucleotide SEQ ID NO: 705
encoding the light chain polypeptide of SEQ ID NO: 706; the
polynucleotide SEQ ID NO: 723 encoding the light chain polypeptide
of SEQ ID NO: 709; the polynucleotide SEQ ID NO: 724 encoding the
heavy chain polypeptide of SEQ ID NO: 19; the polynucleotide SEQ ID
NO: 725 encoding the heavy chain polypeptide of SEQ ID NO: 652; the
polynucleotide SEQ ID NO: 700 encoding the heavy chain polypeptide
of SEQ ID NO: 657; the polynucleotide SEQ ID NO: 663 encoding the
heavy chain polypeptide of SEQ ID NO: 661; the polynucleotide SEQ
ID NO: 703 encoding the heavy chain polypeptide of SEQ ID NO: 704;
the polynucleotide SEQ ID NO: 707 encoding the heavy chain
polypeptide of SEQ ID NO: 708; the polynucleotides of SEQ ID NO:
12, 13, 14, 694 and 695 encoding the complementarity-determining
regions of the aforementioned light chain polypeptides; and the
polynucleotides of SEQ ID NO: 15, 16, 17, 696 and 697 encoding the
complementarity-determining regions of the aforementioned heavy
chain polypeptides, and polynucleotides encoding the variable heavy
and light chain sequences in SEQ ID NO: 657 and SEQ ID NO: 709
respectively, e.g., the nucleic acid sequences in SEQ ID NO: 700
and SEQ ID NO: 723 and fragments or variants thereof, e.g., based
on codon degeneracy. These nucleic acid sequences encoding variable
heavy and light chain sequences may be expressed alone or in
combination and these sequences preferably are fused to suitable
variable constant sequences, e.g., those in SEQ ID NO: 589 and SEQ
ID NO: 587.
[0340] Exemplary nucleotide sequences encoding anti-IL-6 antibodies
of the present subject technology are identified in Table 1. The
polynucleotide sequences shown are to be understood to be
illustrative, rather than limiting. One of skill in the art can
readily determine the polynucleotide sequences that would encode a
given polypeptide and can readily generate coding sequences
suitable for expression in a given expression system, such as by
adapting the polynucleotide sequences provided and/or by generating
them de novo, and can readily produce codon-optimized expression
sequences, for example as described in published U.S. Patent
Application No. 2008/0120732 or using other methods known in the
art.
[0341] In another embodiment of the subject technology,
polynucleotides of the subject technology further comprise, the
following polynucleotide sequence encoding the kappa constant light
chain sequence of SEQ ID NO: 586 which is encoded by the
polynucleotide sequence of SEQ ID NO: 587.
[0342] In another embodiment of the subject technology,
polynucleotides of the subject technology further comprise, the
following polynucleotide sequence encoding the gamma-1 constant
heavy chain polypeptide sequence of SEQ ID NO: 588 which is encoded
by the polynucleotide sequence of SEQ ID NO: 589.
[0343] In one embodiment, the subject technology is directed to an
isolated polynucleotide comprising a polynucleotide encoding an
anti-IL-6 V.sub.H antibody amino acid sequence selected from SEQ ID
NO: 3, 18, 19, 652, 656, 657, 658, 661, 664, 665, 704, and 708 or
encoding a variant thereof wherein at least one framework residue
(FR residue) has been substituted with an amino acid present at the
corresponding position in a rabbit anti-IL-6 antibody V.sub.H
polypeptide or a conservative amino acid substitution. In addition,
the subject technology specifically encompasses humanized anti-IL-6
antibodies or humanized antibody binding fragments or variants
thereof and nucleic acid sequences encoding the foregoing
comprising the humanized variable heavy chain and/or light chain
polypeptides depicted in the sequences contained in FIG. 1-5, or
those identified in Table 1, or variants thereof wherein at least
one framework or CDR residues may be modified. Preferably, if any
modifications are introduced they will not affect adversely the
binding affinity of the resulting anti-IL-6 antibody or fragment or
variant thereof.
[0344] In another embodiment, the subject technology is directed to
an isolated polynucleotide comprising the polynucleotide sequence
encoding an anti-IL-6 V.sub.L antibody amino acid sequence selected
from SEQ ID NO: 2, 20, 647, 651, 660, 666, 699, 702, 706, and 709
or encoding a variant thereof wherein at least one framework
residue (FR residue) has been substituted with an amino acid
present at the corresponding position in a rabbit anti-IL-6
antibody V.sub.L polypeptide or a conservative amino acid
substitution.
[0345] In yet another embodiment, the subject technology is
directed to at least one heterologous polynucleotides comprising a
sequence encoding the polypeptides set forth in SEQ ID NO: 2 and
SEQ ID NO: 3; SEQ ID NO: 2 and SEQ ID NO: 18; SEQ ID NO: 2 and SEQ
ID NO: 19; SEQ ID NO: 20 and SEQ ID NO: 3; SEQ ID NO: 20 and SEQ ID
NO: 18; or SEQ ID NO: 20 and SEQ ID NO: 19.
[0346] In another embodiment, the subject technology is directed to
an isolated polynucleotide that expresses a polypeptide containing
at least one CDR polypeptide derived from an anti-IL-6 antibody
wherein said expressed polypeptide alone specifically binds IL-6 or
specifically binds IL-6 when expressed in association with another
polynucleotide sequence that expresses a polypeptide containing at
least one CDR polypeptide derived from an anti-IL-6 antibody
wherein said at least one CDR is selected from those contained in
the V.sub.L or V.sub.H polypeptides set forth in SEQ ID NO: 3, 18,
19, 652, 656, 657, 658, 661, 664, 665, 704, 708, 2, 20, 647, 651,
660, 666, 699, 702, 706, or 709.
[0347] Host cells and vectors comprising said polynucleotides are
also contemplated.
[0348] In another specific embodiment the subject technology covers
nucleic acid constructs containing any of the foregoing nucleic
acid sequences and combinations thereof as well as recombinant
cells containing these nucleic acid sequences and constructs
containing wherein these nucleic acid sequences or constructs may
be extrachromosomal or integrated into the host cell genome.
[0349] The subject technology further contemplates vectors
comprising the polynucleotide sequences encoding the variable heavy
and light chain polypeptide sequences, as well as the individual
complementarity determining regions (CDRs, or hypervariable
regions) set forth herein, as well as host cells comprising said
sequences. In one embodiment of the subject technology, the host
cell is a yeast cell. In another embodiment of the subject
technology, the yeast host cell belongs to the genus Pichia.
[0350] In some instances, more than one exemplary polynucleotide
encoding a given polypeptide sequence is provided, as summarized in
Table 3.
TABLE-US-00003 TABLE 3 Multiple exemplary polynucleotides encoding
particular polypeptides. Polypeptide SEQ ID NO Exemplary coding SEQ
ID NOs 4 12, 111, 694 5 13, 112, 389, 501 6 14, 113, 695 9 17, 116,
697 39 47, 260 40 48, 261 60 68, 265 72 80, 325, 565, 581 89 97,
134, 166 103 12, 111, 694 104 13, 112, 389, 501 105 14, 113, 695
108 17, 116, 697 126 97, 134, 166 158 97, 134, 166 190 198, 214 191
199, 215 205 213, 469, 485 206 198, 214 207 199, 215 252 47, 260
253 48, 261 257 68, 265 317 80, 325, 565, 581 333 341, 533 381 13,
112, 389, 501 415 423, 439 431 423, 439 461 213, 469, 485 475 483,
499 476 484, 500 477 213, 469, 485 478 486, 502 479 487, 503 480
488, 504 481 489, 505 491 483, 499 492 484, 500 493 13, 112, 389,
501 494 486, 502 495 487, 503 496 488, 504 497 489, 505 525 341,
533 545 553, 585 554 562, 578 556 564, 580 557 80, 325, 565, 581
558 566, 582 570 562, 578 572 564, 580 573 80, 325, 565, 581 574
566, 582 577 553, 585
[0351] In some instances, multiple sequence identifiers refer to
the same polypeptide or polynucleotide sequence, as summarized in
Table 4. References to these sequence identifiers are understood to
be interchangeable, except where context indicates otherwise.
TABLE-US-00004 TABLE 4 Repeated sequences. Each cell lists a group
of repeated sequences included in the sequence listing. SEQ ID NOs
of repeated sequences 4, 103 5, 104, 381, 493 6, 105 9, 108 12, 111
13, 112 14, 113 17, 116 39, 252 40, 253 48, 261 60, 257 68, 265 72,
317, 557, 573 80, 325, 565, 581 89, 126, 158 97, 134, 166 120, 659
190, 206 191, 207 198, 214 199, 215 205, 461, 477 213, 469 333, 525
415, 431 423, 439 475, 491 476, 492 478, 494 479, 495 480, 496 481,
497 483, 499 484, 500 486, 502 487, 503 488, 504 489, 505 545, 577
554, 570 556, 572 558, 574 562, 578 564, 580 566, 582
[0352] Certain exemplary embodiments include polynucleotides that
hybridize under moderately or highly stringent hybridization
conditions to a polynucleotide having one of the exemplary coding
sequences recited in Table 1, and also include polynucleotides that
hybridize under moderately or highly stringent hybridization
conditions to a polynucleotide encoding the same polypeptide as a
polynucleotide having one of the exemplary coding sequences recited
in Table 1, or polypeptide encoded by any of the foregoing
polynucleotides.
[0353] The phrase "high stringency hybridization conditions" refers
to conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. High stringency conditions are sequence
dependent and will be different in different circumstances. Longer
sequences hybridize specifically at higher temperatures. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Probes, "Overview of principles
of hybridization and the strategy of nucleic acid assays" (1993).
Generally, high stringency conditions are selected to be about
5-10.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength pH. The Tm is the
temperature (under defined ionic strength, pH, and nucleic
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). High stringency conditions will be
those in which the salt concentration is less than about 1.0 M
sodium ion, typically about 0.01 to 1.0 M sodium ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides)
and at least about 60.degree. C. for long probes (e.g., greater
than 50 nucleotides). High stringency conditions may also be
achieved with the addition of destabilizing agents such as
formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary high stringency hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C. Such hybridizations and wash steps can be
carried out for, e.g., 1, 2, 5, 10, 15, 30, 60; or more
minutes.
[0354] Nucleic acids that do not hybridize to each other under high
stringency conditions are still substantially related if the
polypeptides that they encode are substantially related. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderate
stringency hybridization conditions. Exemplary "moderate stringency
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. Such hybridizations and wash steps can
be carried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes.
A positive hybridization is at least twice background. Those of
ordinary skill will readily recognize that alternative
hybridization and wash conditions can be utilized to provide
conditions of similar stringency.
[0355] Expression vectors for use in the methods of the subject
technology will further include yeast specific sequences, including
a selectable auxotrophic or drug marker for identifying transformed
yeast strains. A drug marker may further be used to amplify copy
number of the vector in a yeast host cell.
[0356] The polypeptide coding sequence of interest is operably
linked to transcriptional and translational regulatory sequences
that provide for expression of the polypeptide in yeast cells.
These vector components may include, but are not limited to, at
least one of the following: an enhancer element, a promoter, and a
transcription termination sequence. Sequences for the secretion of
the polypeptide may also be included, e.g. a signal sequence, and
the like. A yeast origin of replication is optional, as expression
vectors are often integrated into the yeast genome.
[0357] In one embodiment of the subject technology, the polypeptide
of interest is operably linked, or fused, to sequences providing
for optimized secretion of the polypeptide from yeast diploid
cells.
[0358] Nucleic acids are "operably linked" when placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a signal sequence is operably linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in
the secretion of the polypeptide; a promoter or enhancer is
operably linked to a coding sequence if it affects the
transcription of the sequence. Generally, "operably linked" means
that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading frame.
However, enhancers do not have to be contiguous Linking is
accomplished by ligation at convenient restriction sites or
alternatively via a PCR/recombination method familiar to those
skilled in the art (Gateway.RTM. Technology; Invitrogen, Carlsbad
Calif.). If such sites do not exist, the synthetic oligonucleotide
adapters or linkers are used in accordance with conventional
practice.
[0359] Promoters are untranslated sequences located upstream (5')
to the start codon of a structural gene (generally within about 100
to 1000 bp) that control the transcription and translation of
particular nucleic acid sequences to which they are operably
linked. Such promoters fall into several classes: inducible,
constitutive, and repressible promoters (that increase levels of
transcription in response to absence of a repressor). Inducible
promoters may initiate increased levels of transcription from DNA
under their control in response to some change in culture
conditions, e.g., the presence or absence of a nutrient or a change
in temperature.
[0360] The yeast promoter fragment may also serve as the site for
homologous recombination and integration of the expression vector
into the same site in the yeast genome; alternatively a selectable
marker is used as the site for homologous recombination. Pichia
transformation is described in Cregg, et al. (1985) Mol. Cell.
Biol. 5:3376-3385.
[0361] Examples of suitable promoters from Pichia include the AOX1
and promoter (Cregg, et al. (1989) Mol. Cell. Biol. 9:1316-1323);
ICL1 promoter (Menendez, et al. (2003) Yeast 20(13):1097-108);
glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) (Waterham,
et al. (1997) Gene 186(1):37-44); and FLD1 promoter (Shen, et al.
(1998) Gene 216(1):93-102). The GAP promoter is a strong
constitutive promoter and the AOX and FLD1 promoters are
inducible.
[0362] Other yeast promoters include ADH1, alcohol dehydrogenase
II, GAL4, PHO3, PHO5, Pyk, and chimeric promoters derived
therefrom. Additionally, non-yeast promoters may be used in the
subject technology such as mammalian, insect, plant, reptile,
amphibian, viral, and avian promoters. Most typically the promoter
will comprise a mammalian promoter (potentially endogenous to the
expressed genes) or will comprise a yeast or viral promoter that
provides for efficient transcription in yeast systems.
[0363] The polypeptides of interest may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, e.g. a signal sequence or other
polypeptide having a specific cleavage site at the N-terminus of
the mature protein or polypeptide. In general, the signal sequence
may be a component of the vector, or it may be a part of the
polypeptide coding sequence that is inserted into the vector. The
heterologous signal sequence selected preferably is one that is
recognized and processed through one of the standard pathways
available within the host cell. The S. cerevisiae alpha factor
pre-pro signal has proven effective in the secretion of a variety
of recombinant proteins from P. pastoris. Other yeast signal
sequences include the alpha mating factor signal sequence, the
invertase signal sequence, and signal sequences derived from other
secreted yeast polypeptides. Additionally, these signal peptide
sequences may be engineered to provide for enhanced secretion in
diploid yeast expression systems. Other secretion signals of
interest also include mammalian signal sequences, which may be
heterologous to the protein being secreted, or may be a native
sequence for the protein being secreted. Signal sequences include
pre-peptide sequences, and in some instances may include propeptide
sequences. Many such signal sequences are known in the art,
including the signal sequences found on immunoglobulin chains,
e.g., K28 preprotoxin sequence, PHA-E, FACE, human MCP-1, human
serum albumin signal sequences, human Ig heavy chain, human Ig
light chain, and the like. See Hashimoto, et al. (1998) Protein Eng
11(2): 75; and Kobayashi, et al. (1998) Therapeutic Apheresis 2(4):
257.
[0364] Transcription may be increased by inserting a
transcriptional activator sequence into the vector. These
activators are cis-acting elements of DNA, usually about from 10 to
300 bp, which act on a promoter to increase its transcription.
Transcriptional enhancers are relatively orientation and position
independent, having been found 5' and 3' to the transcription unit,
within an intron, as well as within the coding sequence itself. The
enhancer may be spliced into the expression vector at a position 5'
or 3' to the coding sequence, but is preferably located at a site
5' from the promoter.
[0365] Expression vectors used in eukaryotic host cells may also
contain sequences necessary for the termination of transcription
and for stabilizing the mRNA. Such sequences are commonly available
from 3' to the translation termination codon, in untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA.
[0366] Construction of suitable vectors containing at least one of
the above-listed components employs standard ligation techniques or
PCR/recombination methods. Isolated plasmids or DNA fragments are
cleaved, tailored, and re-ligated in the form desired to generate
the plasmids required or via recombination methods. For analysis to
confirm correct sequences in plasmids constructed, the ligation
mixtures are used to transform host cells, and successful
transformants selected by antibiotic resistance (e.g. ampicillin or
Zeocin.RTM. (phleomycin)) where appropriate. Plasmids from the
transformants are prepared, analyzed by restriction endonuclease
digestion and/or sequenced.
[0367] As an alternative to restriction and ligation of fragments,
recombination methods based on att sites and recombination enzymes
may be used to insert DNA sequences into a vector. Such methods are
described, for example, by Landy (1989) Ann. Rev. Biochem. 58:
913-949; and are known to those of skill in the art. Such methods
utilize intermolecular DNA recombination that is mediated by a
mixture of lambda and E. coli-encoded recombination proteins.
Recombination occurs between specific attachment (att) sites on the
interacting DNA molecules. For a description of att sites see
Weisberg and Landy (1983) Site-Specific Recombination in Phage
Lambda Cold Spring Harbor, N.Y.:Cold Spring Harbor Press), pages
211-250. The DNA segments flanking the recombination sites are
switched, such that after recombination, the att sites are hybrid
sequences comprised of sequences donated by each parental vector.
The recombination can occur between DNAs of any topology.
[0368] Att sites may be introduced into a sequence of interest by
ligating the sequence of interest into an appropriate vector;
generating a PCR product containing att B sites through the use of
specific primers; generating a cDNA library cloned into an
appropriate vector containing att sites.
[0369] The expression host may be further modified by the
introduction of sequences encoding at least one enzymes that
enhance folding and disulfide bond formation, i.e. foldases,
chaperonins, Such sequences may be constitutively or inducibly
expressed in the yeast host cell, using vectors, markers, are known
in the art. Preferably the sequences, including transcriptional
regulatory elements sufficient for the desired pattern of
expression, are stably integrated in the yeast genome through a
targeted methodology.
[0370] For example, the eukaryotic PDI is not only an efficient
catalyst of protein cysteine oxidation and disulfide bond
isomerization, but also exhibits chaperone activity. Co-expression
of PDI can facilitate the production of active proteins having
multiple disulfide bonds. Also of interest is the expression of BIP
(immunoglobulin heavy chain binding protein); cyclophilin; and the
like. In one embodiment of the subject technology, each of the
haploid parental strains expresses a distinct folding enzyme, e.g.
one strain may express BIP, and the other strain may express PDI or
combinations thereof.
[0371] Vectors are used to introduce a foreign substance, such as
DNA, RNA or protein, into an organism or host cell. Typical vectors
include recombinant viruses (for polynucleotides) and liposomes or
other lipid aggregates (for polypeptides and/or polynucleotides). A
"DNA vector" is a replicon, such as plasmid, phage or cosmid, to
which another polynucleotide segment may be attached so as to bring
about the replication of the attached segment. An "expression
vector" is a DNA vector which contains regulatory sequences which
will direct polypeptide synthesis by an appropriate host cell. This
usually means a promoter to bind RNA polymerase and initiate
transcription of mRNA, as well as ribosome binding sites and
initiation signals to direct translation of the mRNA into a
polypeptide(s). Incorporation of a polynucleotide sequence into an
expression vector at the proper site and in correct reading frame,
followed by transformation of an appropriate host cell by the
vector, enables the production of a polypeptide encoded by said
polynucleotide sequence. Exemplary expression vectors and
techniques for their use are described in the following
publications: Old, et al. (1989) Principles of Gene Manipulation:
An Introduction to Genetic Engineering, Blackwell Scientific
Publications, 4th edition; Sambrook, et al. (1989) Molecular
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A
Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory
Press; Gorman, "High Efficiency Gene Transfer into Mammalian
Cells," in DNA Cloning, Volume II, Glover, D. M., Ed., IRL Press,
Washington, D.C., pages 143-190.
[0372] For example, a liposomes or other lipid aggregate may
comprise a lipid such as phosphatidylcholines (lecithins) (PC),
phosphatidylethanolamines (PE), lysolecithins,
lysophosphatidylethanolamines, phosphatidylserines (PS),
phosphatidylglycerols (PG), phosphatidylinositol (PI),
sphingomyelins, cardiolipin, phosphatidic acids (PA), fatty acids,
gangliosides, glucolipids, glycolipids, mono-, di or triglycerides,
ceramides, cerebrosides and combinations thereof; a cationic lipid
(or other cationic amphiphile) such as
1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);
N-cholesteryloxycarbaryl-3,7,12-triazapentadecane-1,15-diamine
(CTAP);
N-[1-(2,3-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammo-
nium bromide (DMRIE);
N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium
bromide (DORIE);
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 3 beta [N-(N',N'-dimethylaminoethane)carbamoly]
cholesterol (DC-Choi); and dimethyldioctadecylammonium (DDAB);
dioleoylphosphatidyl ethanolamine (DOPE), cholesterol-containing
DOPC; and combinations thereof; and/or a hydrophilic polymer such
as polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,
polyethyloxazoline, polyhydroxypropyloxazoline,
polyhydroxypropylmethacrylamide, polymethacrylamide,
polydimethylacrylamide, polyhydroxypropylmethacrylate,
polyhydroxyethylacrylate, hydroxymethylcellulose,
hydroxyethylcellulose, polyethyleneglycol, polyaspartamide and
combinations thereof. Other suitable cationic lipids are described
in Miller (1998) Angewandte Chemie International Edition 37(13-14):
1768-1785 and Cooper, et al. (1998) Chem. Eur. J. 4(1): 137-151.
Liposomes can be crosslinked, partially crosslinked, or free from
crosslinking Crosslinked liposomes can include crosslinked as well
as non-crosslinked components. Suitable cationic liposomes or
cytofectins are commercially available and can also be prepared as
described in Sipkins et al. (1998) Nature Medicine 4(5): 623-626 or
as described in Miller, supra. Exemplary liposomes include a
polymerizable zwitterionic or neutral lipid, a polymerizable
integrin targeting lipid and a polymerizable cationic lipid
suitable for binding a nucleic acid. Liposomes can optionally
include peptides that provide increased efficiency, for example as
described in U.S. Pat. No. 7,297,759. Additional exemplary
liposomes and other lipid aggregates are described in U.S. Pat. No.
7,166,298.
Additional Exemplary Embodiments of the Subject Technology
[0373] In another embodiment, the subject technology contemplates
at least one anti-IL-6 antibodies or antibody fragments or variants
thereof which may specifically bind to the same linear or
conformational epitope(s) and/or compete for binding to the same
linear or conformational epitope(s) on an intact human IL-6
polypeptide or fragment thereof as an anti-IL-6 antibody comprising
Ab1 and chimeric, humanized, single chain antibodies and fragments
thereof (containing at least one CDRs of the afore-identified
antibodies) that specifically bind IL-6, which preferably are
aglycosylated. In a preferred embodiment, the anti-IL-6 antibody or
fragment or variant thereof may specifically bind to the same
linear or conformational epitope(s) and/or compete for binding to
the same linear or conformational epitope(s) on an intact human
IL-6 polypeptide or a fragment thereof as Ab1 and chimeric,
humanized, single chain antibodies and fragments thereof
(containing at least one CDRs of the afore-mentioned antibody) that
specifically bind IL-6, which preferably are aglycosylated.
[0374] In another embodiment of the subject technology, the
anti-IL-6 antibody which may specifically bind to the same linear
or conformational epitopes on an intact IL-6 polypeptide or
fragment thereof that is (are) specifically bound by Ab1 may bind
to an IL-6 epitope(s) ascertained by epitopic mapping using
overlapping linear peptide fragments which span the full length of
the native human IL-6 polypeptide. In one embodiment of the subject
technology, the IL-6 epitope comprises, or alternatively consists
of, at least one residues comprised in IL-6 fragments selected from
those respectively encompassing amino acid residues 37-51, amino
acid residues 70-84, amino acid residues 169-183, amino acid
residues 31-45 and/or amino acid residues 58-72.
[0375] The subject technology is also directed to an anti-IL-6
antibody that binds with the same IL-6 epitope and/or competes with
an anti-IL-6 antibody for binding to IL-6 as an antibody or
antibody fragment disclosed herein, including but not limited to an
anti-IL-6 antibody selected from Ab1 and chimeric, humanized,
single chain antibodies and fragments thereof (containing at least
one CDRs of the afore-mentioned antibody) that specifically bind
IL-6, which preferably are aglycosylated.
[0376] In another embodiment, the subject technology is also
directed to an isolated anti-IL-6 antibody or antibody fragment or
variant thereof comprising at least one of the CDRs contained in
the V.sub.H polypeptide sequences comprising: SEQ ID NO: 3, 18, 19,
22, 38, 54, 70, 86, 102, 117, 118, 123, 139, 155, 171, 187, 203,
219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411,
427, 443, 459, 475, 491, 507, 523, 539, 555, 571, 652, 656, 657,
658, 661, 664, 665, 668, 672, 676, 680, 684, 688, 691, 692, 704, or
708 and/or at least one of the CDRs contained in the V.sub.L
polypeptide sequence consisting of: 2, 20, 21, 37, 53, 69, 85, 101,
119, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298,
314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506,
522, 538, 554, 570, 647, 651, 660, 666, 667, 671, 675, 679, 683,
687, 693, 699, 702, 706, or 709 and the V.sub.H and V.sub.L
sequences depicted in the antibody alignments comprised in FIGS.
8-11 of this application.
[0377] In one embodiment of the subject technology, the anti-IL-6
antibody described herein may comprise at least 2 complementarity
determining regions (CDRs) in each the variable light and the
variable heavy regions which are identical to those contained in an
anti-IL-6 antibody comprising Ab1 and chimeric, humanized, single
chain antibodies and fragments thereof (containing at least one
CDRs of the afore-mentioned antibody) that specifically bind IL-6,
which preferably are aglycosylated.
[0378] In a preferred embodiment, the anti-IL-6 antibody described
herein may comprise at least 2 complementarity determining regions
(CDRs) in each the variable light and the variable heavy regions
which are identical to those contained in Ab1. In another
embodiment, all of the CDRs of the anti-IL-6 antibody discussed
above are identical to the CDRs contained in an anti-IL-6 antibody
comprising Ab1 and chimeric, humanized, single chain antibodies and
fragments thereof (containing at least one CDRs of the
afore-mentioned antibody) that specifically bind IL-6, which
preferably are aglycosylated. In a preferred embodiment of the
subject technology, all of the CDRs of the anti-IL-6 antibody
discussed above are identical to the CDRs contained in Ab1, e.g.,
an antibody comprised of the V.sub.H and V.sub.L sequences
comprised in SEQ ID NO: 657 and SEQ ID NO: 709 respectively.
[0379] The subject technology further contemplates that the one or
more anti-IL-6 antibodies discussed above are aglycosylated or
substantially non-glycosylated (e.g., may contain one or more,
e.g., 1-5 mannose residues); that contain an Fc region that has
been modified to alter effector function, half-life, proteolysis,
and/or glycosylation; are human, humanized, single chain or
chimeric; and are a humanized antibody derived from a rabbit
(parent) anti-IL-6 antibody. Exemplary constant regions that
provide for the production of aglycosylated antibodies in Pichia
are comprised in SEQ ID NO: 588 and SEQ ID NO: 586 which
respectively are encoded by the nucleic acid sequences in SEQ ID
NO: 589 and SEQ ID NO: 587.
[0380] The subject technology further contemplates at least one
anti-IL-6 antibodies wherein the framework regions (FRs) in the
variable light region and the variable heavy regions of said
antibody respectively are human FRs which are unmodified or which
have been modified by the substitution of at most 2 or 3 human FR
residues in the variable light or heavy chain region with the
corresponding FR residues of the parent rabbit antibody, and
wherein said human FRs have been derived from human variable heavy
and light chain antibody sequences which have been selected from a
library of human germline antibody sequences based on their high
level of homology to the corresponding rabbit variable heavy or
light chain regions relative to other human germline antibody
sequences contained in the library.
[0381] In one embodiment of the subject technology, the anti-IL-6
antibody or fragment or variant thereof may specifically bind to
IL-6 expressing human cells and/or to circulating soluble IL-6
molecules in vivo, including IL-6 expressed on or by human cells in
a patient with a disease associated with cells that express
IL-6.
[0382] The subject technology further contemplates anti-IL-6
antibodies or fragments or variants thereof directly or indirectly
attached to a detectable label or therapeutic agent.
[0383] The subject technology also contemplates at least one
nucleic acid sequences which result in the expression of an
anti-IL-6 antibody or antibody fragment or variant thereof as set
forth above, including those comprising, or alternatively
consisting of, yeast or human preferred codons. The subject
technology also contemplates vectors (including plasmids or
recombinant viral vectors) comprising said nucleic acid
sequence(s). The subject technology also contemplates host cells or
recombinant host cells expressing at least one of the antibodies
set forth above, including a mammalian, yeast, bacterial, and
insect cells. In a preferred embodiment, the host cell is a yeast
cell. In a further preferred embodiment, the yeast cell is a
diploidal yeast cell. In a more preferred embodiment, the yeast
cell is a Pichia yeast.
[0384] The subject technology also contemplates a method of
treatment comprising administering to a patient with a disease or
condition associated with psoriatic arthritis a therapeutically
effective amount of at least one anti-IL-6 antibody or
antigen-binding fragment or variant thereof. The diseases that may
be treated are presented in the non-limiting list set forth above.
In another embodiment the treatment further includes the
administration of another therapeutic agent or regimen selected
from chemotherapy, radiotherapy, cytokine administration or gene
therapy agent. For example, drugs associated with the treatment of
psoriatic arthritis including but not limited to TNF-.alpha.
inhibitors, glyococordicoids, triamcinolone, dexamethasone,
prednisone, may also be administered sequentially or subsequently
with at least one anti-IL-6 antibody or antigen-binding fragment or
variant thereof described herein. Further examples of drugs
associated with the treatment of psoriatic arthritis include but
are not limited to ARISTOCORT (triamcinolone), BAYCADROM
(dexamethasone), DECADRON (dexamethasone), DELTASONE (prednisone),
DEXAMETHASONE INTENSOL (dexamethasone), ENBREL (etancercept),
HUMIRA (adalimumab), REMICADE (infliximab), RIDUARA (aruaofin), and
SIMPONI.RTM. (golimumab).
Anti-IL-6 Activity
[0385] As stated previously, IL-6 is a member of a family of
cytokines that promote cellular responses through a receptor
complex consisting of at least one subunit of the
signal-transducing glycoprotein gp130 and the IL-6 receptor
(IL-6R). The IL-6R may also be present in a soluble form (sIL-6R).
IL-6 binds to IL-6R, which then dimerizes the signal-transducing
receptor gp130.
[0386] It is believed that the anti-IL-6 antibodies of the subject
technology, or IL-6 binding fragments or variants thereof, are
useful by exhibiting anti-IL-6 activity. In one non-limiting
embodiment of the subject technology, the anti-IL-6 antibodies of
the subject technology, or IL-6 binding fragments or variants
thereof, exhibit anti-IL-6 activity by binding to IL-6 which may be
soluble IL-6 or cell surface expressed IL-6 and/or may prevent or
inhibit the binding of IL-6 to IL-6R and/or activation
(dimerization) of the gp130 signal-transducing glycoprotein and the
formation of IL-6/IL-6R/gp130 multimers and the biological effects
of any of the foregoing. The subject anti-IL-6 antibodies may
possess different antagonistic activities based on where (i.e.,
epitope) the particular antibody binds IL-6 and/or how it affects
the formation of the foregoing IL-6 complexes and/or multimers and
the biological effects thereof. Consequently, different anti-IL-6
antibodies according to the subject technology e.g., may be better
suited for preventing or treating conditions involving the
formation and accumulation of substantial soluble IL-6 such as
rheumatoid arthritis whereas other antibodies may be favored in
treatments wherein the prevention of IL-6/IL-6R/gp130 or
IL-6/IL-6R/gp130 multimers is a desired therapeutic outcome. This
can be determined in binding and other assays.
[0387] The anti-IL-6 activity of the anti-IL-6 antibody of the
present subject technology, and fragments and variants thereof
having binding specificity to IL-6, may also be described by their
strength of binding or their affinity for IL-6. This also may
affect their therapeutic properties. In one embodiment of the
subject technology, the anti-IL-6 antibodies of the present subject
technology, and fragments thereof having binding specificity to
IL-6, bind to IL-6 with a dissociation constant (K.sub.D) of less
than or equal to 5.times.10.sup.-7, 10.sup.-7, 5.times.10.sup.-8,
10.sup.-8, 5.times.10.sup.-9, 10.sup.-9, 5.times.10.sup.-10,
10.sup.-10, 5.times.10.sup.-11, 10.sup.-11, 5.times.10.sup.-12,
10.sup.-12, 5.times.10.sup.-13, 10.sup.-13, 5.times.10.sup.-14,
10.sup.-14, 5.times.10.sup.-15 or 10.sup.-15. Preferably, the
anti-IL-6 antibodies and fragments and variants thereof bind IL-6
with a dissociation constant of less than or equal to
5.times.10.sup.-10.
[0388] In another embodiment of the subject technology, the
anti-IL-6 activity of the anti-IL-6 antibodies of the present
subject technology, and fragments and variants thereof having
binding specificity to IL-6, bind to IL-6 with an off-rate of less
than or equal to 10.sup.-4 S.sup.-1, 5.times.10.sup.-5 S.sup.-1,
10.sup.-5 S.sup.-1, 5.times.10.sup.-6 S.sup.-1, 10.sup.-6 S.sup.-1,
5.times.10.sup.-7 S.sup.-1, or 10.sup.-7 S.sup.-1. In one
embodiment of the subject technology, the anti-IL-6 antibodies of
the subject technology, and fragments and variants thereof having
binding specificity to IL-6, bind to a linear or conformational
IL-6 epitope.
[0389] In a further embodiment of the subject technology, the
anti-IL-6 activity of the anti-IL-6 antibodies of the present
subject technology, and fragments and variants thereof having
binding specificity to IL-6, exhibit anti-IL-6 activity by
ameliorating or reducing the symptoms of, or alternatively
treating, or preventing, diseases and disorders associated with
IL-6. Non-limiting examples of diseases and disorders associated
with IL-6 are set forth infra. In another embodiment of the subject
technology, the anti-IL-6 antibodies described herein, or IL-6
binding fragments and variants thereof, do not have binding
specificity for IL-6R or the gp-130 signal-transducing
glycoprotein.
B-Cell Screening and Isolation
[0390] In one embodiment, the present subject technology provides
methods of isolating a clonal population of antigen-specific B
cells that may be used for isolating at least one antigen-specific
cell. As described and exemplified infra, these methods contain a
series of culture and selection steps that can be used separately,
in combination, sequentially, repetitively, or periodically.
Preferably, these methods are used for isolating at least one
antigen-specific cell, which can be used to produce a monoclonal
antibody, which is specific to a desired antigen, or a nucleic acid
sequence corresponding to such an antibody.
[0391] In one embodiment, the present subject technology provides a
method comprising the steps of: [0392] (a) preparing a cell
population comprising at least one antigen-specific B cell; [0393]
(b) enriching the cell population, e.g., by chromatography, to form
an enriched cell population comprising at least one
antigen-specific B cell; [0394] (c) isolating a single B cell from
the enriched B cell population; and [0395] (d) determining whether
the single B cell produces an antibody specific to the antigen.
[0396] In another embodiment, the present subject technology
provides an improvement to a method of isolating a single,
antibody-producing B cell, the improvement comprising enriching a B
cell population obtained from a host that has been immunized or
naturally exposed to an antigen, wherein the enriching step
precedes any selection steps, comprises at least one culturing
step, and results in a clonal population of B cells that produces a
single monoclonal antibody specific to said antigen.
[0397] Throughout this application, a "clonal population of B
cells" refers to a population of B cells that only secrete a single
antibody specific to a desired antigen. That is to say that these
cells produce only one type of monoclonal antibody specific to the
desired antigen.
[0398] In the present application, "enriching" a cell population
cells means increasing the frequency of desired cells, typically
antigen-specific cells, contained in a mixed cell population, e.g.,
a B cell-containing isolate derived from a host that is immunized
against a desired antigen. Thus, an enriched cell population
encompasses a cell population having a higher frequency of
antigen-specific cells as a result of an enrichment step, but this
population of cells may contain and produce different
antibodies.
[0399] The general term "cell population" encompasses pre- and a
post-enrichment cell populations, keeping in mind that when
multiple enrichment steps are performed, a cell population can be
both pre- and post-enrichment. For example, in one embodiment, the
present subject technology provides a method: [0400] (a) harvesting
a cell population from an immunized host to obtain a harvested cell
population; [0401] (b) creating at least one single cell suspension
from the harvested cell population; [0402] (c) enriching at least
one single cell suspension to form a first enriched cell
population; [0403] (d) enriching the first enriched cell population
to form a second enriched cell population; [0404] (e) enriching the
second enriched cell population to form a third enriched cell
population; and [0405] (f) selecting an antibody produced by an
antigen-specific cell of the third enriched cell population.
[0406] Each cell population may be used directly in the next step,
or it can be partially or wholly frozen for long- or short- term
storage or for later steps. Also, cells from a cell population can
be individually suspended to yield single cell suspensions. The
single cell suspension can be enriched, such that a single cell
suspension serves as the pre-enrichment cell population. Then, at
least one antigen-specific single cell suspensions together form
the enriched cell population; the antigen-specific single cell
suspensions can be grouped together, e.g., re-plated for further
analysis and/or antibody production.
[0407] In one embodiment, the present subject technology provides a
method of enriching a cell population to yield an enriched cell
population having an antigen-specific cell frequency that is about
50% to about 100%, or increments therein. Preferably, the enriched
cell population has an antigen-specific cell frequency at least
about 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100%.
[0408] In another embodiment, the present subject technology
provides a method of enriching a cell population whereby the
frequency of antigen-specific cells is increased by at least about
2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or increments
therein.
[0409] Throughout this application, the term "increment" is used to
define a numerical value in varying degrees of precision, e.g., to
the nearest 10, 1, 0.1, 0.01. The increment can be rounded to any
measurable degree of precision, and the increment need not be
rounded to the same degree of precision on both sides of a range.
For example, the range 1 to 100 or increments therein includes
ranges such as 20 to 80, 5 to 50, and 0.4 to 98. When a range is
open-ended, e.g., a range of less than 100, increments therein
means increments between 100 and the measurable limit. For example,
less than 100 or increments therein means 0 to 100 or increments
therein unless the feature, e.g., temperature, is not limited by
0.
[0410] Antigen-specificity can be measured with respect to any
antigen. The antigen can be any substance to which an antibody can
bind including, but not limited to, peptides, proteins or fragments
thereof; carbohydrates; organic and inorganic molecules; receptors
produced by animal cells, bacterial cells, and viruses; enzymes;
agonists and antagonists of biological pathways; hormones; and
cytokines Exemplary antigens include, but are not limited to, IL-2,
IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-.alpha., IFN-.gamma.,
BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor
(HGF) and Hepcidin. Preferred antigens include IL-6, IL-13,
TNF-.alpha., VEGF-.alpha., Hepatocyte Growth Factor (HGF) and
Hepcidin. In a method utilizing more than one enrichment step, the
antigen used in each enrichment step can be the same as or
different from one another. Multiple enrichment steps with the same
antigen may yield a large and/or diverse population of
antigen-specific cells; multiple enrichment steps with different
antigens may yield an enriched cell population with
cross-specificity to the different antigens.
[0411] Enriching a cell population can be performed by any
cell-selection means known in the art for isolating
antigen-specific cells. For example, a cell population can be
enriched by chromatographic techniques, e.g., Miltenyi bead or
magnetic bead technology. The beads can be directly or indirectly
attached to the antigen of interest. In a preferred embodiment, the
method of enriching a cell population includes at least one
chromatographic enrichment step.
[0412] A cell population can also be enriched by performed by any
antigen-specificity assay technique known in the art, e.g., an
ELISA assay or a halo assay. ELISA assays include, but are not
limited to, selective antigen immobilization (e.g., biotinylated
antigen capture by streptavidin, avidin, or neutravidin coated
plate), non-specific antigen plate coating, and through an antigen
build-up strategy (e.g., selective antigen capture followed by
binding partner addition to generate a heteromeric protein-antigen
complex). The antigen can be directly or indirectly attached to a
solid matrix or support, e.g., a column. A halo assay comprises
contacting the cells with antigen-loaded beads and labeled
anti-host antibody specific to the host used to harvest the B
cells. The label can be, e.g., a fluorophore. In one embodiment, at
least one assay enrichment step is performed on at least one single
cell suspension. In another embodiment, the method of enriching a
cell population includes at least one chromatographic enrichment
step and at least one assay enrichment step.
[0413] Methods of "enriching" a cell population by size or density
are known in the art. See, e.g., U.S. Pat. No. 5,627,052. These
steps can be used in the present method in addition to enriching
the cell population by antigen-specificity.
[0414] The cell populations of the present subject technology
contain at least one cell capable of recognizing an antigen.
Antigen-recognizing cells include, but are not limited to, B cells,
plasma cells, and progeny thereof. In one embodiment, the present
subject technology provides a clonal cell population containing a
single type of antigen-specific B-cell, i.e., the cell population
produces a single monoclonal antibody specific to a desired
antigen.
[0415] In such embodiment, it is believed that the clonal
antigen-specific population of B cells consists predominantly of
antigen-specific, antibody-secreting cells, which are obtained by
the novel culture and selection protocol provided herein.
Accordingly, the present subject technology also provides methods
for obtaining an enriched cell population containing at least one
antigen-specific, antibody-secreting cell. In one embodiment, the
present subject technology provides an enriched cell population
containing about 50% to about 100%, or increments therein, at least
about 60%, 70%, 80%, 90%, or 100% of antigen-specific,
antibody-secreting cells.
[0416] In one embodiment, the present subject technology provides a
method of isolating a single B cell by enriching a cell population
obtained from a host before any selection steps, e.g., selecting a
particular B cell from a cell population and/or selecting an
antibody produced by a particular cell. The enrichment step can be
performed as one, two, three, or more steps. In one embodiment, a
single B cell is isolated from an enriched cell population before
confirming whether the single B cell secretes an antibody with
antigen-specificity and/or a desired property.
[0417] In one embodiment, a method of enriching a cell population
is used in a method for antibody production and/or selection. Thus,
the present subject technology provides a method comprising
enriching a cell population before selecting an antibody. The
method can include the steps of: preparing a cell population
comprising at least one antigen-specific cell, enriching the cell
population by isolating at least one antigen-specific cell to form
an enriched cell population, and inducing antibody production from
at least one antigen-specific cell. In a preferred embodiment, the
enriched cell population contains more than one antigen-specific
cell. In one embodiment, each antigen-specific cell of the enriched
population is cultured under conditions that yield a clonal
antigen-specific B cell population before isolating an antibody
producing cell therefrom and/or producing an antibody using said B
cell, or a nucleic acid sequence corresponding to such an antibody.
In contrast to prior techniques where antibodies are produced from
a cell population with a low frequency of antigen-specific cells,
the present subject technology allows antibody selection from among
a high frequency of antigen-specific cells. Because an enrichment
step is used prior to antibody selection, the majority of the
cells, preferably virtually all of the cells, used for antibody
production are antigen-specific. By producing antibodies from a
population of cells with an increased frequency of antigen
specificity, the quantity and variety of antibodies are
increased.
[0418] In the antibody selection methods of the present subject
technology, an antibody is preferably selected after an enrichment
step and a culture step that results in a clonal population of
antigen-specific B cells. The methods can further comprise a step
of sequencing a selected antibody or portions thereof from at least
one isolated, antigen-specific cells. Any method known in the art
for sequencing can be employed and can include sequencing the heavy
chain, light chain, variable region(s), and/or complementarity
determining region(s) (CDR).
[0419] In addition to the enrichment step, the method for antibody
selection can also include at least one steps of screening a cell
population for antigen recognition and/or antibody functionality.
For example, the desired antibodies may have specific structural
features, such as binding to a particular epitope or mimicry of a
particular structure; antagonist or agonist activity; or
neutralizing activity, e.g., inhibiting binding between the antigen
and a ligand. In one embodiment, the antibody functionality screen
is ligand-dependent. Screening for antibody functionality includes,
but is not limited to, an in vitro protein-protein interaction
assay that recreates the natural interaction of the antigen ligand
with recombinant receptor protein; and a cell-based response that
is ligand dependent and easily monitored (e.g., proliferation
response). In one embodiment, the method for antibody selection
includes a step of screening the cell population for antibody
functionality by measuring the inhibitory concentration (IC50). In
one embodiment, at least one of the isolated, antigen-specific
cells produces an antibody having an IC50 of less than about 100,
50, 30, 25, 10 .mu.g/mL, or increments therein.
[0420] In addition to the enrichment step, the method for antibody
selection can also include at least one steps of screening a cell
population for antibody binding strength. Antibody binding strength
can be measured by any method known in the art (e.g.,
Biacore.RTM.). In one embodiment, at least one of the isolated,
antigen-specific cells produces an antibody having a high antigen
affinity, e.g., a dissociation constant (Kd) of less than about
5.times.10.sup.-10 M-1, preferably about 1.times.10.sup.-13 to
5.times.10.sup.-10, 1.times.10.sup.-12 to 1.times.10.sup.-10,
1.times.10.sup.-12 to 7.5.times.10.sup.-11, 1.times.10.sup.-11,
2.times.10.sup.-11, about 1.5.times.10.sup.-11 or less, or
increments therein. In this embodiment, the antibodies are said to
be affinity mature. In a preferred embodiment, the affinity of the
antibodies is comparable to or higher than the affinity of any one
of Panorex.RTM. (edrecolomab), Rituxan.RTM. (rituximab),
Herceptin.RTM. (traztuzumab), Mylotarg.RTM. (gentuzumab),
Campath.RTM. (alemtuzumab), Zevalin.RTM. (ibritumomab),
Erbitux.RTM. (cetuximab), Avastin.RTM. (bevicizumab), Raptiva.RTM.
(efalizumab), Remicade.RTM. (infliximab), Humira.RTM. (adalimumab),
and Xolair.RTM. (omalizumab). Preferably, the affinity of the
antibodies is comparable to or higher than the affinity of
Humira.RTM.. The affinity of an antibody can also be increased by
known affinity maturation techniques. In one embodiment, at least
one cell population is screened for at least one of, preferably
both, antibody functionality and antibody binding strength.
[0421] In addition to the enrichment step, the method for antibody
selection can also include at least one steps of screening a cell
population for antibody sequence homology, especially human
homology. In one embodiment, at least one of the isolated,
antigen-specific cells produces an antibody that has a homology to
a human antibody of at least about 50% to about 100%, or increments
therein, or at least about 60%, 70%, 80%, 85%, 90%, or 95%
homologous. The antibodies can be humanized to increase the
homology to a human sequence by techniques known in the art such as
CDR grafting or selectivity determining residue grafting (SDR).
[0422] In another embodiment, the present subject technology also
provides the antibodies themselves according to any of the
embodiments described above in terms of IC50, Kd, and/or
homology.
Methods of Humanizing Antibodies
[0423] In another embodiment of the subject technology, there is
provided a method for humanizing antibody heavy and light chains.
In this embodiment, the following method is followed for the
humanization of the heavy and light chains:
[0424] Light Chain
[0425] 1. Identify the amino acid that is the first one following
the signal peptide sequence. This is the start of Framework 1. The
signal peptide starts at the first initiation methionine and is
typically, but not necessarily 22 amino acids in length for rabbit
light chain protein sequences. The start of the mature polypeptide
can also be determined experimentally by N-terminal protein
sequencing, or can be predicted using a prediction algorithm. This
is also the start of Framework 1 as classically defined by those in
the field.
Example
RbtV.sub.L Amino acid residue 1 in FIG. 1, starting `AYDM . . . `
(SEQ ID NO: 733)
[0426] 2. Identify the end of Framework
[0427] 3. This is typically 86-90 amino acids following the start
of Framework 1 and is typically a cysteine residue preceded by two
tyrosine residues. This is the end of the Framework 3 as
classically defined by those in the field.
Example
RbtV.sub.L amino acid residue 88 in FIG. 1, ending as `TYYC` (SEQ
ID NO: 733)
[0428] 3. Use the rabbit light chain sequence of the polypeptide
starting from the beginning of Framework 1 to the end of Framework
3 as defined above and perform a sequence homology search for the
most similar human antibody protein sequences. This will typically
be a search against human germline sequences prior to antibody
maturation in order to reduce the possibility of immunogenicity,
however any human sequences can be used. Typically a program like
BLAST can be used to search a database of sequences for the most
homologous. Databases of human antibody sequences can be found from
various sources such as NCBI (National Center for Biotechnology
Information).
Example
[0429] RbtV.sub.L amino acid sequence from residues numbered 1
through 88 in FIG. 1 is BLASTed against a human antibody germline
database. The top three unique returned sequences are shown in FIG.
1 as L12A (SEQ ID NO: 734), V1 (SEQ ID NO: 735), and Vx02 (SEQ ID
NO: 736).
[0430] 4. Generally the most homologous human germline variable
light chain sequence is then used as the basis for humanization.
However those skilled in the art may decide to use another sequence
that wasn't the highest homology as determined by the homology
algorithm, based on other factors including sequence gaps and
framework similarities.
Example
[0431] In FIG. 1, L12A (SEQ ID NO: 734) was the most homologous
human germline variable light chain sequence and is used as the
basis for the humanization of RbtV.sub.L.
[0432] 5. Determine the framework and CDR arrangement (FR1, FR2,
FR3, CDR1 & CDR2) for the human homolog being used for the
light chain humanization. This is using the traditional layout as
described in the field. Align the rabbit variable light chain
sequence with the human homolog, while maintaining the layout of
the framework and CDR regions.
Example
[0433] In FIG. 1, the RbtV.sub.L sequence is aligned with the human
homologous sequence L12A, and the framework and CDR domains are
indicated.
[0434] 6. Replace the human homologous light chain sequence CDR1
and CDR2 regions with the CDR1 and CDR2 sequences from the rabbit
sequence. If there are differences in length between the rabbit and
human CDR sequences then use the entire rabbit CDR sequences and
their lengths. It is possible that the specificity, affinity and/or
immunogenicity of the resulting humanized antibody may be unaltered
if smaller or larger sequence exchanges are performed, or if
specific residue(s) are altered, however the exchanges as described
have been used successfully, but do not exclude the possibility
that other changes may be permitted.
Example
[0435] In FIG. 1, the CDR1 and CDR2 amino acid residues of the
human homologous variable light chain L12A are replaced with the
CDR1 and CDR2 amino acid sequences from the RbtV.sub.L rabbit
antibody light chain sequence. The human L12A frameworks 1, 2 and 3
are unaltered. The resulting humanized sequence is shown below as
V.sub.Lh from residues numbered 1 through 88. Note that the only
residues that are different from the L12A human sequence are
underlined, and are thus rabbit-derived amino acid residues. In
this example only 8 of the 88 residues are different than the human
sequence.
[0436] 7. After framework 3 of the new hybrid sequence created in
Step 6, attach the entire CDR3 of the rabbit light chain antibody
sequence. The CDR3 sequence can be of various lengths, but is
typically 9 to 15 amino acid residues in length. The CDR3 region
and the beginning of the following framework 4 region are defined
classically and identifiable by those skilled in the art. Typically
the beginning of Framework 4, and thus after the end of CDR3
consists of the sequence `FGGG . . . ` (SEQ ID NO: 743), however
some variation may exist in these residues.
Example
[0437] In FIG. 1, the CDR3 of RbtV.sub.L (amino acid residues
numbered 89-100) is added after the end of framework 3 in the
humanized sequence indicated as V.sub.Lh.
[0438] 8. The rabbit light chain framework 4, which is typically
the final 11 amino acid residues of the variable light chain and
begins as indicated in Step 7 above and typically ends with the
amino acid sequence ` . . . VVKR` (SEQ ID NO: 744) is replaced with
the nearest human light chain framework 4 homolog, usually from
germline sequence. Frequently this human light chain framework 4 is
of the sequence `FGGGTKVEIKR` (SEQ ID NO: 745). It is possible that
other human light chain framework 4 sequences that are not the most
homologous or otherwise different may be used without affecting the
specificity, affinity and/or immunogenicity of the resulting
humanized antibody. This human light chain framework 4 sequence is
added to the end of the variable light chain humanized sequence
immediately following the CDR3 sequence from Step 7 above. This is
now the end of the variable light chain humanized amino acid
sequence.
Example
[0439] In FIG. 1, Framework 4 (FR4) of the RbtV.sub.L rabbit light
chain sequence is shown above a homologous human FR4 sequence. The
human FR4 sequence is added to the humanized variable light chain
sequence (V.sub.Lh) right after the end of the CD3 region added in
Step 7 above.
[0440] In addition, FIGS. 8 and 9 depict preferred humanized
anti-IL-6 variable heavy and variable light chain sequences
humanized from the variable heavy and light regions in Ab1
according to the subject technology. These humanized light and
heavy chain regions are respectively contained in the polypeptides
set forth in SEQ ID NO: 647, or 651 and in SEQ ID NO: 652, 656, 657
or 658. The CDR2 of the humanized variable heavy region in SEQ ID
NO: 657 (containing a serine substitution in CDR2) is set forth in
SEQ ID NO: 658. Alignments illustrating variants of the light and
heavy chains are shown in FIGS. 10 and 11, respectively, with
sequence differences within the CDR regions highlighted. Sequence
identifiers of CDR sequences and of exemplary coding sequences are
summarized in Table 1, above.
[0441] Heavy Chain
[0442] 1. Identify the amino acid that is the first one following
the signal peptide sequence. This is the start of Framework 1. The
signal peptide starts at the first initiation methionine and is
typically 19 amino acids in length for rabbit heavy chain protein
sequences. Typically, but not necessarily always, the final 3 amino
acid residues of a rabbit heavy chain signal peptide are ` . . .
VQC`, followed by the start of Framework 1. The start of the mature
polypeptide can also be determined experimentally by N-terminal
protein sequencing, or can be predicted using a prediction
algorithm. This is also the start of Framework 1 as classically
defined by those in the field.
Example
RbtV.sub.H Amino acid residue 1 in FIG. 1, starting `QEQL . . . `
(SEQ ID NO: 738)
[0443] 2. Identify the end of Framework 3. This is typically 95-100
amino acids following the start of Framework 1 and typically has
the final sequence of ` . . . CAR` (although the alanine can also
be a valine). This is the end of the Framework 3 as classically
defined by those in the field.
Example
RbtV.sub.H amino acid residue 98 in FIG. 1, ending as ` . . . FCVR`
(SEQ ID NO: 738)
[0444] 3. Use the rabbit heavy chain sequence of the polypeptide
starting from the beginning of Framework 1 to the end of Framework
3 as defined above and perform a sequence homology search for the
most similar human antibody protein sequences. This will typically
be against a database of human germline sequences prior to antibody
maturation in order to reduce the possibility of immunogenicity,
however any human sequences can be used. Typically a program like
BLAST can be used to search a database of sequences for the most
homologous. Databases of human antibody sequences can be found from
various sources such as NCBI (National Center for Biotechnology
Information).
Example
[0445] RbtV.sub.H amino acid sequence from residues numbered 1
through 98 in FIG. 1 is BLASTed against a human antibody germline
database. The top three unique returned sequences are shown in FIG.
1 as 3-64-04 (SEQ ID NO: 739), 3-66-04 (SEQ ID NO: 740), and
3-53-02 (SEQ ID NO: 741).
[0446] 4. Generally the most homologous human germline variable
heavy chain sequence is then used as the basis for humanization.
However those skilled in the art may decide to use another sequence
that wasn't the most homologous as determined by the homology
algorithm, based on other factors including sequence gaps and
framework similarities.
Example
[0447] 3-64-04 in FIG. 1 was the most homologous human germline
variable heavy chain sequence and is used as the basis for the
humanization of RbtV.sub.H.
[0448] 5. Determine the framework and CDR arrangement (FR1, FR2,
FR3, CDR1 & CDR2) for the human homolog being used for the
heavy chain humanization. This is using the traditional layout as
described in the field. Align the rabbit variable heavy chain
sequence with the human homolog, while maintaining the layout of
the framework and CDR regions.
Example
[0449] In FIG. 1, the RbtV.sub.H sequence is aligned with the human
homologous sequence 3-64-04, and the framework and CDR domains are
indicated.
[0450] 6. Replace the human homologous heavy chain sequence CDR1
and CDR2 regions with the CDR1 and CDR2 sequences from the rabbit
sequence. If there are differences in length between the rabbit and
human CDR sequences then use the entire rabbit CDR sequences and
their lengths. In addition, it may be necessary to replace the
final three amino acids of the human heavy chain Framework 1 region
with the final three amino acids of the rabbit heavy chain
Framework 1. Typically but not always, in rabbit heavy chain
Framework 1 these three residues follow a Glycine residue preceded
by a Serine residue. In addition, it may be necessary replace the
final amino acid of the human heavy chain Framework 2 region with
the final amino acid of the rabbit heavy chain Framework 2.
Typically, but not necessarily always, this is a Glycine residue
preceded by an Isoleucine residue in the rabbit heavy chain
Framework 2. It is possible that the specificity, affinity and/or
immunogenicity of the resulting humanized antibody may be unaltered
if smaller or larger sequence exchanges are performed, or if
specific residue(s) are altered, however the exchanges as described
have been used successfully, but do not exclude the possibility
that other changes may be permitted. For example, a tryptophan
amino acid residue typically occurs four residues prior to the end
of the rabbit heavy chain CDR2 region, whereas in human heavy chain
CDR2 this residue is typically a Serine residue. Changing this
rabbit tryptophan residue to a the human Serine residue at this
position has been demonstrated to have minimal to no effect on the
humanized antibody's specificity or affinity, and thus further
minimizes the content of rabbit sequence-derived amino acid
residues in the humanized sequence.
Example
[0451] In FIG. 1, The CDR1 and CDR2 amino acid residues of the
human homologous variable heavy chain are replaced with the CDR1
and CDR2 amino acid sequences from the RbtV.sub.H rabbit antibody
light chain sequence, except for the boxed residue, which is
tryptophan in the rabbit sequence (position number 63) and Serine
at the same position in the human sequence, and is kept as the
human Serine residue. In addition to the CDR1 and CDR2 changes, the
final three amino acids of Framework 1 (positions 28-30) as well as
the final residue of Framework 2 (position 49) are retained as
rabbit amino acid residues instead of human. The resulting
humanized sequence is shown below as V.sub.Hh from residues
numbered 1 through 98. Note that the only residues that are
different from the 3-64-04 human sequence are underlined, and are
thus rabbit-derived amino acid residues. In this example only 15 of
the 98 residues are different than the human sequence.
[0452] 7. After framework 3 of the new hybrid sequence created in
Step 6, attach the entire CDR3 of the rabbit heavy chain antibody
sequence. The CDR3 sequence can be of various lengths, but is
typically 5 to 19 amino acid residues in length. The CDR3 region
and the beginning of the following framework 4 region are defined
classically and are identifiable by those skilled in the art.
Typically the beginning of framework 4, and thus after the end of
CDR3 consists of the sequence WGXG . . . (where X is usually Q or
P) (SEQ ID NO: 746), however some variation may exist in these
residues.
Example
[0453] The CDR3 of RbtV.sub.H (amino acid residues numbered 99-110)
is added after the end of framework 3 in the humanized sequence
indicated as V.sub.Hh.
[0454] 8. The rabbit heavy chain framework 4, which is typically
the final 11 amino acid residues of the variable heavy chain and
begins as indicated in Step 7 above and typically ends with the
amino acid sequence ` . . . TVSS` (SEQ ID NO: 747) is replaced with
the nearest human heavy chain framework 4 homolog, usually from
germline sequence. Frequently this human heavy chain framework 4 is
of the sequence `WGQGTLVTVSS` (SEQ ID NO: 748). It is possible that
other human heavy chain framework 4 sequences that are not the most
homologous or otherwise different may be used without affecting the
specificity, affinity and/or immunogenicity of the resulting
humanized antibody. This human heavy chain framework 4 sequence is
added to the end of the variable heavy chain humanized sequence
immediately following the CDR3 sequence from Step 7 above. This is
now the end of the variable heavy chain humanized amino acid
sequence.
Example
[0455] In FIG. 1, framework 4 (FR4) of the RbtV.sub.H rabbit heavy
chain sequence is shown above a homologous human heavy FR4
sequence. The human FR4 sequence is added to the humanized variable
heavy chain sequence (V.sub.Hh) right after the end of the CD3
region added in Step 7 above.
Methods of Producing Antibodies and Fragments Thereof
[0456] The subject technology is also directed to the production of
the antibodies described herein or fragments thereof. Recombinant
polypeptides corresponding to the antibodies described herein or
fragments thereof are secreted from polyploidal, preferably diploid
or tetraploid strains of mating competent yeast. In an exemplary
embodiment, the subject technology is directed to methods for
producing these recombinant polypeptides in secreted form for
prolonged periods using cultures comprising polyploid yeast, i.e.,
at least several days to a week, more preferably at least a month
or several months, and even more preferably at least 6 months to a
year or longer. These polyploid yeast cultures will express at
least 10-25 mg/liter of the polypeptide, more preferably at least
50-250 mg/liter, still more preferably at least 500-1000 mg/liter,
and most preferably a gram per liter or more of the recombinant
polypeptide(s).
[0457] In one embodiment of the subject technology a pair of
genetically marked yeast haploid cells are transformed with
expression vectors comprising subunits of a desired
heteromultimeric protein. One haploid cell comprises a first
expression vector, and a second haploid cell comprises a second
expression vector. In another embodiment diploid yeast cells will
be transformed with at least one expression vectors that provide
for the expression and secretion of at least one of the recombinant
polypeptides. In still another embodiment a single haploid cell may
be transformed with at least one vectors and used to produce a
polyploidal yeast by fusion or mating strategies. In yet another
embodiment a diploid yeast culture may be transformed with at least
one vectors providing for the expression and secretion of a desired
polypeptide or polypeptides. These vectors may comprise vectors
e.g., linearized plasmids or other linear DNA products that
integrate into the yeast cell's genome randomly, through homologous
recombination, or using a recombinase such as Cre/Lox or Flp/Frt.
Optionally, additional expression vectors may be introduced into
the haploid or diploid cells; or the first or second expression
vectors may comprise additional coding sequences; for the synthesis
of heterotrimers; heterotetramers. The expression levels of the
non-identical polypeptides may be individually calibrated, and
adjusted through appropriate selection, vector copy number,
promoter strength and/or induction and the like. The transformed
haploid cells are genetically crossed or fused. The resulting
diploid or tetraploid strains are utilized to produce and secrete
fully assembled and biologically functional proteins, humanized
antibodies described herein or fragments thereof.
[0458] The use of diploid or tetraploid cells for protein
production provides for unexpected benefits. The cells can be grown
for production purposes, i.e. scaled up, and for extended periods
of time, in conditions that can be deleterious to the growth of
haploid cells, which conditions may include high cell density;
growth in minimal media; growth at low temperatures; stable growth
in the absence of selective pressure; and which may provide for
maintenance of heterologous gene sequence integrity and maintenance
of high level expression over time. Without wishing to be bound
thereby, the inventors theorize that these benefits may arise, at
least in part, from the creation of diploid strains from two
distinct parental haploid strains. Such haploid strains can
comprise numerous minor autotrophic mutations, which mutations are
complemented in the diploid or tetraploid, enabling growth and
enhanced production under highly selective conditions.
[0459] Transformed mating competent haploid yeast cells provide a
genetic method that enables subunit pairing of a desired protein.
Haploid yeast strains are transformed with each of two expression
vectors, a first vector to direct the synthesis of one polypeptide
chain and a second vector to direct the synthesis of a second,
non-identical polypeptide chain. The two haploid strains are mated
to provide a diploid host where optimized target protein production
can be obtained.
[0460] Optionally, additional non-identical coding sequence(s) are
provided. Such sequences may be present on additional expression
vectors or in the first or the second expression vectors. As is
known in the art, multiple coding sequences may be independently
expressed from individual promoters; or may be coordinately
expressed through the inclusion of an "internal ribosome entry
site" or "IRES", which is an element that promotes direct internal
ribosome entry to the initiation codon, such as ATG, of a cistron
(a protein encoding region), thereby leading to the cap-independent
translation of the gene. IRES elements functional in yeast are
described by Thompson, et al. (2001) PNAS 98: 12866-12868.
[0461] In one embodiment of the subject technology, antibody
sequences are produced in combination with a secretory J chain,
which provides for enhanced stability of IgA. See U.S. Pat. Nos.
5,959,177 and 5,202,422.
[0462] In a preferred embodiment the two haploid yeast strains are
each auxotrophic, and require supplementation of media for growth
of the haploid cells. The pair of auxotrophs are complementary,
such that the diploid product will grow in the absence of the
supplements required for the haploid cells. Many such genetic
markers are known in yeast, including requirements for amino acids
(e.g. met, lys, his, arg), nucleosides (e.g. ura3, ade1); and the
like. Amino acid markers may be preferred for the methods of the
subject technology. Alternatively diploid cells which contain the
desired vectors can be selected by other means, e.g., by use of
other markers, such as green fluorescent protein, antibiotic
resistance genes, various dominant selectable markers, and the
like.
[0463] Two transformed haploid cells may be genetically crossed and
diploid strains arising from this mating event selected by their
hybrid nutritional requirements and/or antibiotic resistance
spectra. Alternatively, populations of the two transformed haploid
strains are spheroplasted and fused, and diploid progeny
regenerated and selected. By either method, diploid strains can be
identified and selectively grown based on their ability to grow in
different media than their parents. For example, the diploid cells
may be grown in minimal medium that may include antibiotics. The
diploid synthesis strategy has certain advantages. Diploid strains
have the potential to produce enhanced levels of heterologous
protein through broader complementation to underlying mutations,
which may impact the production and/or secretion of recombinant
protein. Furthermore, once stable strains have been obtained, any
antibiotics used to select those strains do not necessarily need to
be continuously present in the growth media.
[0464] As noted above, in some embodiments a haploid yeast may be
transformed with a single or multiple vectors and mated or fused
with a non-transformed cell to produce a diploid cell containing
the vector or vectors. In other embodiments, a diploid yeast cell
may be transformed with at least one vectors that provide for the
expression and secretion of a desired heterologous polypeptide by
the diploid yeast cell.
[0465] In one embodiment of the subject technology, two haploid
strains are transformed with a library of polypeptides, e.g. a
library of antibody heavy or light chains. Transformed haploid
cells that synthesize the polypeptides are mated with the
complementary haploid cells. The resulting diploid cells are
screened for functional protein. The diploid cells provide a means
of rapidly, conveniently and inexpensively bringing together a
large number of combinations of polypeptides for functional
testing. This technology is especially applicable for the
generation of heterodimeric protein products, where optimized
subunit synthesis levels are critical for functional protein
expression and secretion.
[0466] In another embodiment of the subject technology, the
expression level ratio of the two subunits is regulated in order to
maximize product generation. Heterodimer subunit protein levels
have been shown previously to impact the final product generation.
Simmons (2002) J Immunol Methods. 263(1-2): 133-47. Regulation can
be achieved prior to the mating step by selection for a marker
present on the expression vector. By stably increasing the copy
number of the vector, the expression level can be increased. In
some cases, it may be desirable to increase the level of one chain
relative to the other, so as to reach a balanced proportion between
the subunits of the polypeptide. Antibiotic resistance markers are
useful for this purpose, e.g. Zeocin.RTM. (phleomycin) resistance
marker, G418 resistance and provide a means of enrichment for
strains that contain multiple integrated copies of an expression
vector in a strain by selecting for transformants that are
resistant to higher levels of Zeocin.RTM. (phleomycin) or G418. The
proper ratio (e.g. 1:1; 1:2) of the subunit genes may be important
for efficient protein production. Even when the same promoter is
used to transcribe both subunits, many other factors contribute to
the final level of protein expressed and therefore, it can be
useful to increase the number of copies of one encoded gene
relative to the other. Alternatively, diploid strains that produce
higher levels of a polypeptide, relative to single copy vector
strains, are created by mating two haploid strains, both of which
have multiple copies of the expression vectors.
[0467] Host cells are transformed with the above-described
expression vectors, mated to form diploid strains, and cultured in
conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants or amplifying the genes encoding
the desired sequences. A number of minimal media suitable for the
growth of yeast are known in the art. Any of these media may be
supplemented as necessary with salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as phosphate,
HEPES), nucleosides (such as adenosine and thymidine), antibiotics,
trace elements, and glucose or an equivalent energy source. Any
other necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to the ordinarily skilled artisan.
[0468] Secreted proteins are recovered from the culture medium. A
protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF)
may be useful to inhibit proteolytic degradation during
purification, and antibiotics may be included to prevent the growth
of adventitious contaminants. The composition may be concentrated,
filtered, dialyzed, using methods known in the art.
[0469] The diploid cells of the subject technology are grown for
production purposes. Such production purposes desirably include
growth in minimal media, which media lacks pre-formed amino acids
and other complex biomolecules, e.g., media comprising ammonia as a
nitrogen source, and glucose as an energy and carbon source, and
salts as a source of phosphate, calcium and the like. Preferably
such production media lacks selective agents such as antibiotics,
amino acids, purines, pyrimidines The diploid cells can be grown to
high cell density, for example at least about 50 g/L; more usually
at least about 100 g/L; and may be at least about 300, about 400,
about 500 g/L or more.
[0470] In one embodiment of the subject technology, the growth of
the subject cells for production purposes is performed at low
temperatures, which temperatures may be lowered during log phase,
during stationary phase, or both. The term "low temperature" refers
to temperatures of at least about 15.degree. C., more usually at
least about 17.degree. C., and may be about 20.degree. C., and is
usually not more than about 25.degree. C., more usually not more
than about 22.degree. C. In another embodiment of the subject
technology, the low temperature is usually not more than about
28.degree. C. Growth temperature can impact the production of
full-length secreted proteins in production cultures, and
decreasing the culture growth temperature can strongly enhance the
intact product yield. The decreased temperature appears to assist
intracellular trafficking through the folding and
post-translational processing pathways used by the host to generate
the target product, along with reduction of cellular protease
degradation.
[0471] The methods of the subject technology provide for expression
of secreted, active protein, preferably a mammalian protein. In one
embodiment, secreted, "active antibodies", as used herein, refers
to a correctly folded multimer of at least two properly paired
chains, which accurately binds to its cognate antigen. Expression
levels of active protein are usually at least about 10-50 mg/liter
culture, more usually at least about 100 mg/liter, preferably at
least about 500 mg/liter, and may be 1000 mg/liter or more.
[0472] The methods of the subject technology can provide for
increased stability of the host and heterologous coding sequences
during production. The stability is evidenced, for example, by
maintenance of high levels of expression of time, where the
starting level of expression is decreased by not more than about
20%, usually not more than 10%, and may be decreased by not more
than about 5% over about 20 doublings, 50 doublings, 100 doublings,
or more.
[0473] The strain stability also provides for maintenance of
heterologous gene sequence integrity over time, where the sequence
of the active coding sequence and requisite transcriptional
regulatory elements are maintained in at least about 99% of the
diploid cells, usually in at least about 99.9% of the diploid
cells, and preferably in at least about 99.99% of the diploid cells
over about 20 doublings, 50 doublings, 100 doublings, or more.
Preferably, substantially all of the diploid cells maintain the
sequence of the active coding sequence and requisite
transcriptional regulatory elements.
[0474] Other methods of producing antibodies are well known to
those of ordinary skill in the art. For example, methods of
producing chimeric antibodies are now well known in the art. See,
e.g., U.S. Pat. No. 4,816,567; Morrison, et al. (1984) P.N.A.S. USA
81: 8651-55; Neuberger, et al. (1985) Nature 314: 268-270;
Boulianne, et al. (1984) Nature 312: 643-46.
[0475] Likewise, other methods of producing humanized antibodies
are now well known in the art. See, e.g., U.S. Pat. Nos. 5,225,539;
5,530,101; 5,585,089; 5,693,762; 6,054,297; 6,180,370; 6,407,213;
6,548,640; 6,632,927; and 6,639,055; Jones, et al. (1986) Nature
321: 522-525; Reichmann, et al. (1988) Nature 332: 323-327;
Verhoeyen, et al. (1988) Science 239: 1534-36.
[0476] Antibody polypeptides of the subject technology having IL-6
binding specificity may also be produced by constructing, using
conventional techniques well known to those of ordinary skill in
the art, an expression vector containing an operon and a DNA
sequence encoding an antibody heavy chain in which the DNA sequence
encoding the CDRs required for antibody specificity is derived from
a non-human cell source, preferably a rabbit B-cell source, while
the DNA sequence encoding the remaining parts of the antibody chain
is derived from a human cell source.
[0477] A second expression vector is produced using the same
conventional means well known to those of ordinary skill in the
art, said expression vector containing an operon and a DNA sequence
encoding an antibody light chain in which the DNA sequence encoding
the CDRs required for antibody specificity is derived from a
non-human cell source, preferably a rabbit B-cell source, while the
DNA sequence encoding the remaining parts of the antibody chain is
derived from a human cell source.
[0478] The expression vectors are transfected into a host cell by
convention techniques well known to those of ordinary skill in the
art to produce a transfected host cell, said transfected host cell
cultured by conventional techniques well known to those of ordinary
skill in the art to produce said antibody polypeptides.
[0479] The host cell may be co-transfected with the two expression
vectors described above, the first expression vector containing DNA
encoding an operon and a light chain-derived polypeptide and the
second vector containing DNA encoding an operon and a heavy
chain-derived polypeptide. The two vectors contain different
selectable markers, but preferably achieve substantially equal
expression of the heavy and light chain polypeptides.
Alternatively, a single vector may be used, the vector including
DNA encoding both the heavy and light chain polypeptides. The
coding sequences for the heavy and light chains may comprise
cDNA.
[0480] The host cells used to express the antibody polypeptides may
be either a bacterial cell such as E. coli, or a eukaryotic cell.
In a particularly preferred embodiment of the subject technology, a
mammalian cell of a well-defined type for this purpose, such as a
myeloma cell or a Chinese hamster ovary (CHO) cell line may be
used.
[0481] The general methods by which the vectors may be constructed,
transfection methods required to produce the host cell and
culturing methods required to produce the antibody polypeptides
from said host cells all include conventional techniques. Although
preferably the cell line used to produce the antibody is a
mammalian cell line, any other suitable cell line, such as a
bacterial cell line such as an E. coli-derived bacterial strain, or
a yeast cell line, may alternatively be used.
[0482] Similarly, once produced the antibody polypeptides may be
purified according to standard procedures in the art, such as for
example cross-flow filtration, ammonium sulphate precipitation,
affinity column chromatography and the like.
[0483] The antibody polypeptides described herein may also be used
for the design and synthesis of either peptide or non-peptide
mimetics that would be useful for the same therapeutic applications
as the antibody polypeptides of the subject technology. See, for
example, Saragobi et al. (1991) Science 253: 792-795.
Exemplary Embodiments of Heavy and Light Chain Polypeptides and
Polynucleotides
[0484] This section recites exemplary embodiments of heavy and
light chain polypeptides, as well as exemplary polynucleotides
encoding such polypeptides. These exemplary polynucleotides are
suitable for expression in the disclosed Pichia expression
system.
[0485] In certain embodiments, the present subject technology
encompasses polynucleotides having at least about 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity (sequence homology) to the polynucleotides recited in this
application or that encode polypeptides recited in this
application, or that hybridize to said polynucleotides under
conditions of low-stringency, moderate-stringency, or
high-stringency conditions, preferably those that encode
polypeptides (e.g. an immunoglobulin heavy and light chain, a
single-chain antibody, an antibody fragment) that have at least one
of the biological activities set forth herein, including without
limitation thereto specific binding to an IL-6 polypeptide. In
another aspect, the subject technology encompasses a composition
comprising such a polynucleotide and/or a polypeptide encoded by
such a polynucleotide. In yet another aspect, the subject
technology encompasses a method of treatment of a disease or
condition associated with IL-6 or that may be prevented, treated,
or ameliorated with an IL-6 antagonist such as Ab1 (e.g. psoriatic
arthritis) comprising administration of a composition comprising
such a polynucleotide and/or polypeptide.
[0486] In certain preferred embodiments, a heavy chain polypeptide
will comprise at least one of the CDR sequences of the heavy and/or
light chain polypeptides recited herein (including those contained
in the heavy and light chain polypeptides recited herein) and at
least one of the framework region polypeptides recited herein,
including those depicted in FIGS. 1 and 8-11 or Table 1, and
contained in the heavy and light chain polypeptide sequences
recited herein. In certain preferred embodiments, a heavy chain
polypeptide will comprise at least one Framework 4 region sequences
as depicted in FIGS. 1 and 8-11 or Table 1, or as contained in a
heavy or light chain polypeptide recited herein.
[0487] In certain preferred embodiments, a light chain polypeptide
will comprise at least one of the CDR sequences of the heavy and/or
light chain polypeptides recited herein (including those contained
in the heavy and light chain polypeptides recited herein) and at
least one of the Framework region polypeptides recited herein,
including those depicted in FIGS. 1 and 8-11 or Table 1, and
contained in the heavy and light chain polypeptide sequences
recited herein. In certain preferred embodiments, a light chain
polypeptide will comprise at least one Framework 4 region sequences
as depicted in FIGS. 1 and 8-11 or Table 1, or as contained in a
heavy or light chain polypeptide recited herein.
[0488] In any of the embodiments recited herein, certain of the
sequences recited may be substituted for each other, unless the
context indicates otherwise. The recitation that particular
sequences may be substituted for one another, where such
recitations are made, are understood to be illustrative rather than
limiting, and it is also understood that such substitutions are
encompassed even when no illustrative examples of substitutions are
recited, For example, wherever at least one of the Ab1 light chain
polypeptides is recited, e.g. any of SEQ ID NO: 2, 20, 647, 651,
660, 666, 699, 702, 706, or 709, another Ab1 light chain
polypeptide may be substituted unless the context indicates
otherwise. Similarly, wherever one of the Ab1 heavy chain
polypeptides is recited, e.g. any of SEQ ID NO: 3, 18, 19, 652,
656, 657, 658, 661, 664, 665, 704, or 708, another Ab1 heavy chain
polypeptide may be substituted unless the context indicates
otherwise. Likewise, wherever one of the Ab1 light chain
polynucleotides is recited, e.g. any of SEQ ID NO: 10, 662, 698,
701, or 705, another Ab1 light chain polynucleotide may be
substituted unless the context indicates otherwise. Similarly,
wherever one of the Ab1 heavy chain polynucleotides is recited,
e.g. any of SEQ ID NO: 11, 663, 700, 703, or 707, another Ab1 heavy
chain polynucleotide may be substituted unless the context
indicates otherwise.
[0489] Additionally, recitation of any member of any of the
following groups is understood to encompass substitution by any
other member of the group, as follows: Ab2 Light chain polypeptides
(SEQ ID NO: 21 and 667); Ab2 Light chain polynucleotides (SEQ ID
NO: 29 and 669); Ab2 Heavy chain polypeptides (SEQ ID NO: 22 and
668); Ab2 Heavy chain polynucleotides (SEQ ID NO: 30 and 670); Ab3
Light chain polypeptides (SEQ ID NO: 37 and 671); Ab3 Light chain
polynucleotides (SEQ ID NO: 45 and 673); Ab3 Heavy chain
polypeptides (SEQ ID NO: 38 and 672); Ab3 Heavy chain
polynucleotides (SEQ ID NO: 46 and 674); Ab4 Light chain
polypeptides (SEQ ID NO: 53 and 675); Ab4 Light chain
polynucleotides (SEQ ID NO: 61 and 677); Ab4 Heavy chain
polypeptides (SEQ ID NO: 54 and 676); Ab4 Heavy chain
polynucleotides (SEQ ID NO: 62 and 678); Ab5 Light chain
polypeptides (SEQ ID NO: 69 and 679); Ab5 Light chain
polynucleotides (SEQ ID NO: 77 and 681); Ab5 Heavy chain
polypeptides (SEQ ID NO: 70 and 680); Ab5 Heavy chain
polynucleotides (SEQ ID NO: 78 and 682); Ab6 Light chain
polypeptides (SEQ ID NO: 85 and 683); Ab6 Light chain
polynucleotides (SEQ ID NO: 93 and 685); Ab6 Heavy chain
polypeptides (SEQ ID NO: 86 and 684); Ab6 Heavy chain
polynucleotides (SEQ ID NO: 94 and 686); Ab7 Light chain
polypeptides (SEQ ID NO: 101, 119, 687, 693); Ab7 Light chain
polynucleotides (SEQ ID NO: 109 and 689); Ab7 Heavy chain
polypeptides (SEQ ID NO: 102, 117, 118, 688, 691, and 692); Ab7
Heavy chain polynucleotides (SEQ ID NO: 110 and 690); Ab1 Light
Chain CDR1 polynucleotides (SEQ ID NO: 12 and 694); Ab1 Light Chain
CDR3 polynucleotides (SEQ ID NO: 14 and 695); Ab1 Heavy Chain CDR2
polynucleotides (SEQ ID NO: 16 and 696) and Ab1 Heavy Chain CDR3
polynucleotides (SEQ ID NO: 17 and 697). Exemplary Ab1-encoding
polynucleotide sequences include but are not limited to SEQ ID NO:
662, 663, 698, 700, 701, 703, 705, 707, 720, 721, 722, 723, 724,
and 725.
Screening Assays
[0490] The subject technology also includes screening assays
designed to assist in the identification of diseases and disorders
associated with IL-6 in patients exhibiting symptoms of an IL-6
associated disease or disorder, especially psoriatic arthritis.
[0491] In one embodiment of the subject technology, the anti-IL-6
antibodies of the subject technology, or IL-6 binding fragments or
variants thereof, are used to detect the presence of IL-6 in a
biological sample obtained from a patient exhibiting symptoms of a
disease or disorder associated with IL-6. The presence of IL-6, or
elevated levels thereof when compared to pre-disease levels of IL-6
in a comparable biological sample, may be beneficial in diagnosing
a disease or disorder associated with IL-6.
[0492] Another embodiment of the subject technology provides a
diagnostic or screening assay to assist in diagnosis of diseases or
disorders associated with IL-6 in patients exhibiting symptoms of
an IL-6 associated disease or disorder identified herein,
comprising assaying the level of IL-6 expression in a biological
sample from said patient using a post-translationally modified
anti-IL-6 antibody or binding fragment or variant thereof. The
anti-IL-6 antibody or binding fragment or variant thereof may be
post-translationally modified to include a detectable moiety such
as set forth previously in the disclosure.
[0493] The IL-6 level in the biological sample is determined using
a modified anti-IL-6 antibody or binding fragment or variant
thereof as set forth herein, and comparing the level of IL-6 in the
biological sample against a standard level of IL-6 (e.g., the level
in normal biological samples). The skilled clinician would
understand that some variability may exist between normal
biological samples, and would take that into consideration when
evaluating results.
[0494] The above-recited assay may also be useful in monitoring a
disease or disorder, where the level of IL-6 obtained in a
biological sample from a patient believed to have an IL-6
associated disease or disorder is compared with the level of IL-6
in prior biological samples from the same patient, in order to
ascertain whether the IL-6 level in said patient has changed with,
for example, a treatment regimen. A skilled clinician would
understand that a biological sample includes, but is not limited
to, sera, plasma, urine, saliva, mucous, pleural fluid, synovial
fluid and spinal fluid.
Labels
[0495] As stated above, antibodies and fragments and variants
thereof may be modified post-translationally to add effector
moieties such as chemical linkers, detectable moieties such as for
example fluorescent dyes, enzymes, substrates, bioluminescent
materials, radioactive materials, and chemiluminescent moieties, or
functional moieties such as for example streptavidin, avidin,
biotin, a cytotoxin, a cytotoxic agent, and radioactive
materials.
[0496] The anti-IL-6 antibodies and antigen-binding fragments
thereof described herein may be modified post-translationally to
add effector moieties such as chemical linkers, detectable moieties
such as for example fluorescent dyes, enzymes, substrates,
bioluminescent materials, radioactive materials, chemiluminescent
moieties, a cytotoxic agent, radioactive materials, or functional
moieties.
[0497] A wide variety of entities, e.g., ligands, may be coupled to
the oligonucleotides as known in the art. Ligands may include
naturally occurring molecules, or recombinant or synthetic
molecules. Exemplary ligands include, but are not limited to,
avadin, biotin, peptides, peptidomimetics, polylysine (PLL),
polyethylene glycol (PEG), mPEG, cationic groups, spermine,
spermidine, polyamine, thyrotropin, melanotropin, lectin,
glycoprotein, surfactant protein A, mucin, glycosylated
polyaminoacids, transferrin, aptamer, immunoglobulins (e.g.,
antibodies), insulin, transferrin, albumin, sugar, lipophilic
molecules (e.g., steroids, bile acids, cholesterol, cholic acid,
and fatty acids), vitamin A, vitamin E, vitamin K, vitamin B, folic
acid, B12, riboflavin, biotin, pyridoxal, vitamin cofactors,
lipopolysaccharide, hormones and hormone receptors, lectins,
carbohydrates, multivalent carbohydrates, radiolabeled markers,
fluorescent dyes, and derivatives thereof. See, e.g., U.S. Pat.
Nos. 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437;
6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; and 6,559,
279.
[0498] Additionally, moieties may be added to the antigen or
epitope to increase half-life in vivo (e.g., by lengthening the
time to clearance from the blood stream. Such techniques include,
for example, adding PEG moieties (also termed pegylation), and are
well-known in the art. See U.S. Patent Application Publication No.
2003/0031671.
[0499] An anti-IL-6 antibody or antigen binding fragment thereof,
described herein may be "attached" to a substrate when it is
associated with the solid label through a non-random chemical or
physical interaction. The attachment may be through a covalent
bond. However, attachments need not be covalent or permanent.
Materials may be attached to a label through a "spacer molecule" or
"linker group." Such spacer molecules are molecules that have a
first portion that attaches to the biological material and a second
portion that attaches to the label. Thus, when attached to the
label, the spacer molecule separates the label and the biological
materials, but is attached to both. Methods of attaching biological
material (e.g., label) to a label are well known in the art, and
include but are not limited to chemical coupling.
Detectable Labels
[0500] The anti-IL-6 antibody or antigen-binding fragments
described herein may be modified post-translationally to add
effector labels such as chemical linkers, detectable labels such as
for example fluorescent dyes, enzymes, substrates, bioluminescent
materials, radioactive materials, and chemiluminescent labels, or
functional labels such as for example streptavidin, avidin, biotin,
a cytotoxin, a cytotoxic agent, and radioactive materials. Further
exemplary enzymes include, but are not limited to, horseradish
peroxidase, acetylcholinesterase, alkaline phosphatase,
.beta.-galactosidase and luciferase. Further exemplary fluorescent
materials include, but are not limited to, rhodamine, fluorescein,
fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine,
phycoerythrin and dansyl chloride. Further exemplary
chemiluminescent labels include, but are not limited to, luminol.
Further exemplary bioluminescent materials include, but are not
limited to, luciferin and aequorin. Further exemplary radioactive
materials include, but are not limited to, bismuth-213
(.sup.213Bs), carbon-14 (.sup.14C), carbon-11 (.sup.11C),
chlorine-18 (Cl.sup.18), chromium-51 (.sup.51Cr), cobalt-57
(.sup.57Co), cobalt-60 (.sup.60Co), copper-64 (.sup.64Cu),
copper-67 (.sup.67Cu), dysprosium-165 (.sup.165Dy), erbium-169
(.sup.169Er), fluorine-18 (.sup.18F), gallium-67 (.sup.67Ga),
gallium-68 (.sup.68Ga), germanium-68 (.sup.68Ge), holmium-166
(.sup.166Ho), indium-111 (.sup.111In), iodine-125 (.sup.125I),
iodine-123 (.sup.124I), iodine-124 (.sup.124I), iodine-131
(.sup.131I), iridium-192 (.sup.192Ir), iron-59 (.sup.59Fe),
krypton-81 (.sup.81Kr), lead-212 (.sup.212Pb), lutetium-177
(.sup.177Lu), molybdenum-99 (.sup.99Mo), nitrogen-13 (.sup.13N),
oxygen-15 (.sup.15O), palladium-103 (.sup.103Pd), phosphorus-32
(.sup.32P), potassium-42 (.sup.42K), rhenium-186 (.sup.186Re),
rhenium-188 (.sup.188Re), rubidium-81 (.sup.81Rb), rubidium-82
(.sup.82Rb), samarium-153 (.sup.153Sm), selenium-75 (.sup.75Se),
sodium-24 (.sup.24Na), strontium-82 (.sup.82Sr), strontium-89
(.sup.89Sr), sulfur 35 (.sup.35S), technetium-99m (.sup.99Tc),
thallium-201 (.sup.201Tl), tritium (.sup.3H), xenon-133
(.sup.133Xe), ytterbium-169 (.sup.169Yb), ytterbium-177
(.sup.177Yb), and yttrium-90 (.sup.90Y).
Cytotoxic Agents
[0501] The anti-IL-6 antibodies and antigen-binding fragments
described herein may be conjugated to cytotoxic agents including,
but are not limited to, methotrexate, aminopterin,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
dacarbazine; alkylating agents such as mechlorethamine, thiotepa
chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine
(CCNU), 1-methylnitrosourea, cyclophosphamide, mechlorethamine,
busulfan, dibromomannitol, streptozotocin, mitomycin C,
cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin
(paraplatin); anthracyclines include daunorubicin (formerly
daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin,
idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics
include dactinomycin (actinomycin D), bleomycin, calicheamicin,
mithramycin, and anthramycin (AMC); and antimitotic agents such as
the vinca alkaloids, vincristine and vinblastine. Other cytotoxic
agents include paclitaxel (TAXOL.RTM.), ricin, pseudomonas
exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium
bromide, emetine, etoposide, teniposide, colchicine, dihydroxy
anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, puromycin, procarbazine,
hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)),
interferons, and mixtures of these cytotoxic agents.
[0502] Further cytotoxic agents include, but are not limited to,
chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,
gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin
C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF
antagonists, EGFR antagonists, platins, taxols, irinotecan,
5-fluorouracil, gemcitabine, leucovorin, steroids,
cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine,
vincristine, vindesine and vinorelbine), mustines, tyrosine kinase
inhibitors, radiotherapy, sex hormone antagonists, selective
androgen receptor modulators, selective estrogen receptor
modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists,
interleukins (e.g. IL-12 or IL-2), IL-12R antagonists,
Erbitux.RTM., Avastin.RTM., Pertuzumab, anti-CD20 antibodies,
Rituxan.RTM., ocrelizumab, ofatumumab, DXL625, Herceptin.RTM., or
any combination thereof. Toxic enzymes from plants and bacteria
such as ricin, diphtheria toxin and Pseudomonas toxin may be
conjugated to the humanized antibodies, or binding fragments
thereof, to generate cell-type-specific-killing reagents. Youle, et
al. (1980) Proc. Nat'l Acad. Sci. USA 77: 5483; Gilliland, et al.
(1980) Proc. Nat'l Acad. Sci. USA 77: 4539; Krolick, et al. (1980)
Proc. Nat'l Acad. Sci. USA 77: 5419. Other cytotoxic agents include
cytotoxic ribonucleases. See U.S. Pat. No. 6,653,104.
[0503] The anti-IL-6 antibodies and antigen-binding fragments
described herein may be conjugated to a radionuclide that emits
alpha or beta particles (e.g., radioimmunoconjuagtes). Such
radioactive isotopes include but are not limited to beta-emitters
such as phosphorus-32 (.sup.32P), scandium-47 (.sup.47Sc),
copper-67 (.sup.67Cu), gallium-67 (.sup.67Ga), yttrium-88
(.sup.88Y), yttrium-90 (.sup.90Y), iodine-125 (.sup.125I),
iodine-131 (.sup.131I), samarium-153 (.sup.153Sm), lutetium-177
(.sup.177Lu), rhenium-186 (.sup.186Re), rhenium-188 (.sup.188Re),
and alpha-emitters such as astatine-211 (.sup.211At), lead-212
(.sup.212Pb), bismuth-212 (.sup.212Bi), bismuth-213 (.sup.213Bi) or
actinium-225 (.sup.225Ac).
[0504] Methods are known in the art for conjugating an anti-IL-6
antibody described herein to a label, such as those methods
described by Hunter, et al. (1962) Nature 144: 945; David, et al.
(1974) Biochemistry 13: 1014; Pain, et al. (1981) J. Immunol. Meth.
40: 219; and Nygren (1982) Histochem and Cytochem 30: 407.
Substrates
[0505] The anti-IL-6 antibodies and antigen-binding fragments
thereof described herein may be attached to a substrate. A number
of substrates (e.g., solid supports) known in the art are suitable
for use with the anti-IL-6 antibody described herein. The substrate
may be modified to contain channels or other configurations. See
Fung (2004) [Ed.] Protein Arrays: Methods and Protocols Humana
Press and Kambhampati (2004) [Ed.] Protein Microarray Technology
John Wiley & Sons.
[0506] Substrate materials include, but are not limited to
acrylics, agarose, borosilicate glass, carbon (e.g., carbon
nanofiber sheets or pellets), cellulose acetate, cellulose,
ceramics, gels, glass (e.g., inorganic, controlled-pore, modified,
soda-lime, or functionalized glass), latex, magnetic beads,
membranes, metal, metalloids, nitrocellulose, NYLON.RTM., optical
fiber bundles, organic polymers, paper, plastics,
polyacryloylmorpholide, poly(4-methylbutene), poly(ethylene
terephthalate), poly(vinyl butyrate), polyacrylamide, polybutylene,
polycarbonate, polyethylene, polyethyleneglycol terephthalate,
polyformaldehyde, polymethacrylate, polymethylmethacrylate,
polypropylene, polysaccharides, polystyrene, polyurethanes,
polyvinylacetate, polyvinylchloride, polyvinylidene difluoride
(PVDF), polyvinylpyrrolidinone, rayon, resins, rubbers,
semiconductor materials, SEPHAROSE.RTM., silica, silicon, styrene
copolymers, TEFLON.RTM., and variety of other polymers.
[0507] Substrates need not be flat and can include any type of
shape including spherical shapes (e.g., beads) or cylindrical
shapes (e.g., fibers). Materials attached to solid supports may be
attached to any portion of the solid support (e.g., may be attached
to an interior portion of a porous solid support material).
[0508] The substrate body may be in the form of a bead, box,
column, cylinder, disc, dish (e.g., glass dish, PETRI dish), fiber,
film, filter, microtiter plate (e.g., 96-well microtiter plate),
multi-bladed stick, net, pellet, plate, ring, rod, roll, sheet,
slide, stick, tray, tube, or vial. The substrate may be a singular
discrete body (e.g., a single tube, a single bead), any number of a
plurality of substrate bodies (e.g, a rack of 10 tubes, several
beads), or combinations thereof (e.g., a tray comprises a plurality
of microtiter plates, a column filled with beads, a microtiter
plate filed with beads).
[0509] An anti-IL-6 antibody or antigen-binding fragment thereof
may be "attached" to a substrate when it is associated with the
solid substrate through a non-random chemical or physical
interaction. The attachment may be through a covalent bond.
However, attachments need not be covalent or permanent. Materials
may be attached to a substrate through a "spacer molecule" or
"linker group." Such spacer molecules are molecules that have a
first portion that attaches to the biological material and a second
portion that attaches to the substrate. Thus, when attached to the
substrate, the spacer molecule separates the substrate and the
biological materials, but is attached to both. Methods of attaching
biological material (e.g., label) to a substrate are well known in
the art, and include but are not limited to chemical coupling.
[0510] Plates, such as microtiter plates, which support and contain
the solid-phase for solid-phase synthetic reactions may be used.
Microtiter plates may house beads that are used as the solid-phase.
By "particle" or "microparticle" or "nanoparticle" or "bead" or
"microbead" or "microsphere" herein is meant microparticulate
matter having any of a variety of shapes or sizes. The shape may be
generally spherical but need not be spherical, being, for example,
cylindrical or polyhedral. As will be appreciated by those in the
art, the particles may comprise a wide variety of materials
depending on their use, including, but not limited to, cross-linked
starch, dextrans, cellulose, proteins, organic polymers including
styrene polymers such as polystyrene and methylstyrene as well as
other styrene co-polymers, plastics, glass, ceramics, acrylic
polymers, magnetically responsive materials, colloids, thoriasol,
carbon graphite, titanium dioxide, nylon, latex, and TEFLON.RTM..
See e.g., "Microsphere Detection Guide" from Bangs Laboratories,
Fishers, Ind.
[0511] The anti-IL-6 antibody or antigen-binding fragment may be
attached to on any of the forms of substrates described herein
(e.g., bead, box, column, cylinder, disc, dish (e.g., glass dish,
PETRI dish), fiber, film, filter, microtiter plate (e.g., 96-well
microtiter plate), multi-bladed stick, net, pellet, plate, ring,
rod, roll, sheet, slide, stick, tray, tube, or vial). In
particular, particles or beads may be a component of a gelling
material or may be separate components such as latex beads made of
a variety of synthetic plastics (e.g., polystyrene). The label
(e.g., streptavidin) may be bound to a substrate (e.g., bead).
Assessment of Inflammatory Markers
[0512] Known inflammatory markers (e.g., IL-6) may be measured to
assess the risk for psoriatic arthritis or the severity of
psoriatic arthritis. These markers may be measured from serum,
synovial fluid, or skin biopsies using known methods in the art
(e.g., immunoassays).
IL-6 Serum Levels
[0513] Serum IL-6 levels may be measured as a pharmacodynamic
marker evaluate the effect of neutralization of IL-6 levels. Serum
IL-6 levels may be measured using an immunoassay (e.g., ELISA
assay). A decrease of serum IL-6 levels may be indicative of a
lessening of inflammation.
Serum Inflammatory Biomarkers
[0514] Serum biomarkers may be measured to determine the expression
of pro-inflammatory cytokines and other soluble biomarkers that may
correlate with psoriatic arthritis (PsA) disease activity including
but not limited to acute phase reactants, serum pro-inflammatory
cytokines (e.g., IL-1, TNF-.alpha., IFN-.gamma., IL-12p40, IL-17),
chemokines (e.g., RANTES, MIP-1.alpha., MCP-1), matrix
metalloproteinases (e.g., MMP-2, MMP-3, MMP-9) and other biomarkers
associated with inflammation and autoimmune pathways that are known
in the art. Soluble biomarkers of bone and cartilage metabolism
(e.g., osteocalcin and other collagen degradation products) may
also be assessed by an immunoassay (e.g., ELISA). A decrease in a
serum inflammatory biomarker may be indicative of a lessening of
inflammation.
Immunohistochemistry of Skin Biopsies
[0515] Skin biopsies may be collected for biomarker analysis
including whole genome array analysis and immunohistochemistry
(IHC). Immunohistochemical analysis may include the measurement of
epidermal thickness, frequency of resident and inflammatory cell
populations (e.g., T cells, macrophages, and keratinocytes) and
other inflammatory markers related to the IL-6 pathway known in the
art. Specifically, the following specific antigens may be assessed
per standard IHC procedure using the formalin-fixed samples: CD3,
CD68, keratin 16, FoxP3, IL-6R and MMP-3. A decrease in an
inflammatory biomarker in a skin biopsy may be indicative of a
lessening of inflammation.
Administration
[0516] In one embodiment of the subject technology, the anti-IL-6
antibodies described herein, or IL-6 binding fragments or variants
thereof, as well as combinations of said antibody fragments or
variants, are administered to a subject at a concentration of
between about 0.1 and 20 mg/kg, such as about 0.4 mg/kg, about 0.8
mg/kg, about 1.6 mg/kg, or about 4 mg/kg, of body weight of
recipient subject. For example, compositions comprising the
anti-IL-6 antibodies described herein may comprise at least about
0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,
280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,
410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg. In a
preferred embodiment of the subject technology, the anti-IL-6
antibodies described herein, or IL-6 binding fragments or variants
thereof, as well as combinations of said antibody fragments or
variants, are administered to a subject at a concentration of about
0.4 mg/kg of body weight of recipient subject. In a preferred
embodiment of the subject technology, the anti-IL-6 antibodies
described herein, or IL-6 binding fragments or variants thereof, as
well as combinations of said antibody fragments or variants, are
administered to a recipient subject with a frequency of once every
twenty-six weeks or less, such as once every sixteen weeks or less,
once every eight weeks or less, or once every four weeks, or less.
In another preferred embodiment of the subject technology, the
anti-IL-6 antibodies described herein, or IL-6 binding fragments or
variants thereof, as well as combinations thereof, are administered
to a recipient subject with a frequency at most once per period of
approximately one week, such as at most once per period of
approximately two weeks, such as at most once per period of
approximately four weeks, such as at most once per period of
approximately eight weeks, such as at most once per period of
approximately twelve weeks, such as at most once per period of
approximately sixteen weeks, such as at most once per period of
approximately twenty-four weeks.
[0517] The compositions described herein may be administered in any
of the following routes: buccal, epicutaneous, epidural, infusion,
inhalation, intraarterial, intracardial, intracerebroventricular,
intradermal, intramuscular, intranasal, intraocular,
intraperitoneal, intraspinal, intrathecal, intravenous, oral,
parenteral, pulmonary, rectally via an enema or suppository,
subcutaneous, subdermal, sublingual, transdermal, and transmucosal.
The preferred routes of administration are intravenous injection or
infusion. The administration can be local, where the composition is
administered directly, close to, in the locality, near, at, about,
or in the vicinity of, the site(s) of disease, e.g., local (joint)
or systemic, wherein the composition is given to the patient and
passes through the body widely, thereby reaching the site(s) of
disease. Local administration (e.g., subcutaneous injection) may be
accomplished by administration to the cell, tissue, organ, and/or
organ system, which encompasses and/or is affected by the disease,
and/or where the disease signs and/or symptoms are active or are
likely to occur (e.g., swollen joint). Administration can be
topical with a local effect, composition is applied directly where
its action is desired (e.g., joint). Further, administration of a
composition comprising an effective amount of an anti-IL-6 antibody
selected from the group consisting of Ab1-Ab36 or an
antigen-binding fragment thereof, may be subcutaneous.
[0518] For each of the recited embodiments, the compounds can be
administered by a variety of dosage forms as known in the art. Any
biologically-acceptable dosage form known to persons of ordinary
skill in the art, and combinations thereof, are contemplated.
Examples of such dosage forms include, without limitation, chewable
tablets, quick dissolve tablets, effervescent tablets,
reconstitutable powders, elixirs, liquids, solutions, suspensions,
emulsions, tablets, multi-layer tablets, bi-layer tablets,
capsules, soft gelatin capsules, hard gelatin capsules, caplets,
lozenges, chewable lozenges, beads, powders, gum, granules,
particles, microparticles, dispersible granules, cachets, douches,
suppositories, creams, topicals, inhalants, aerosol inhalants,
patches, particle inhalants, implants, depot implants, ingestibles,
injectables (including subcutaneous, intramuscular, intravenous,
and intradermal), infusions, and combinations thereof.
[0519] Other compounds which can be included by admixture are, for
example, medically inert ingredients (e.g., solid and liquid
diluent), such as lactose, dextrosesaccharose, cellulose, starch or
calcium phosphate for tablets or capsules, olive oil or ethyl
oleate for soft capsules and water or vegetable oil for suspensions
or emulsions; lubricating agents such as silica, talc, stearic
acid, magnesium or calcium stearate and/or polyethylene glycols;
gelling agents such as colloidal clays; thickening agents such as
gum tragacanth or sodium alginate, binding agents such as starches,
arabic gums, gelatin, methylcellulose, carboxymethylcellulose or
polyvinylpyrrolidone; disintegrating agents such as starch, alginic
acid, alginates or sodium starch glycolate; effervescing mixtures;
dyestuff; sweeteners; wetting agents such as lecithin, polysorbates
or laurylsulphates; and other therapeutically acceptable accessory
ingredients, such as humectants, preservatives, buffers and
antioxidants, which are known additives for such formulations.
[0520] Liquid dispersions for oral administration can be syrups,
emulsions, solutions, or suspensions. The syrups can contain as a
carrier, for example, saccharose or saccharose with glycerol and/or
mannitol and/or sorbitol. The suspensions and the emulsions can
contain a carrier, for example a natural gum, agar, sodium
alginate, pectin, methylcellulose, carboxymethylcellulose, or
polyvinyl alcohol.
[0521] In further embodiments, the present subject technology
provides kits including at least one container comprising
pharmaceutical dosage units comprising an effective amount of at
least one antibody and fragments thereof of the present subject
technology. Kits may include instructions, directions, labels,
marketing information, warnings, or information pamphlets.
Dosages
[0522] The amount of anti-IL-6 antibodies in a therapeutic
composition according to any embodiments of this subject technology
may vary according to factors such as the disease state, age,
gender, weight, patient history, risk factors, predisposition to
disease, administration route, pre-existing treatment regime (e.g.,
possible interactions with other medications), and weight of the
individual. Dosage regimens may be adjusted to provide the optimum
therapeutic response. For example, a single bolus may be
administered, several divided doses may be administered over time,
or the dose may be proportionally reduced or increased as indicated
by the exigencies of therapeutic situation.
[0523] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of antibodies, or antigen-binding fragments
thereof, calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the subject technology
are dictated by and directly dependent on the unique
characteristics of the antibodies, and fragments thereof, and the
particular therapeutic effect to be achieved, and the limitations
inherent in the art of compounding such an antibodies, and
fragments thereof, for the treatment of sensitivity in individuals.
In therapeutic use for treatment of conditions in mammals (e.g.,
humans) for which the antibodies and fragments thereof of the
present subject technology or an appropriate pharmaceutical
composition thereof are effective, the antibodies and fragments
thereof of the present subject technology may be administered in an
effective amount. The dosages as suitable for this subject
technology may be a composition, a pharmaceutical composition or
any other compositions described herein.
[0524] The dosage may be administered as a single dose, a double
dose, a triple dose, a quadruple dose, and/or a quintuple dose. The
dosages may be administered singularly, simultaneously, and
sequentially. For example, two doses may be administered on the
same day followed by subsequent two doses four weeks later.
[0525] The dosage form may be any form of release known to persons
of ordinary skill in the art. The compositions of the present
subject technology may be formulated to provide immediate release
of the active ingredient or sustained or controlled release of the
active ingredient. In a sustained release or controlled release
preparation, release of the active ingredient may occur at a rate
such that blood levels are maintained within a therapeutic range
but below toxic levels over an extended period of time (e.g., 4 to
24 hours). The preferred dosage forms include immediate release,
extended release, pulse release, variable release, controlled
release, timed release, sustained release, delayed release, long
acting, and combinations thereof, and are known in the art.
[0526] It will be appreciated that the pharmacological activity of
the compositions may be monitored using standard pharmacological
models that are known in the art. Furthermore, it will be
appreciated that the compositions comprising an anti-IL-6
antibodies or antigen-binding fragments thereof may be incorporated
or encapsulated in a suitable polymer matrix or membrane for
site-specific delivery, or may be functionalized with specific
targeting agents capable of effecting site specific delivery. These
techniques, as well as other drug delivery techniques are well
known in the art. Determination of optimal dosages for a particular
situation is within the capabilities of those skilled in the art.
See, e.g., Grennaro (2005) [Ed.] Remington: The Science and
Practice of Pharmacy [21.sup.st Ed.]
[0527] In another embodiment of the subject technology, the
anti-IL-6 antibodies described herein, or IL-6 binding fragments or
variants thereof, as well as combinations of said antibody
fragments or variants, are administered to a subject in a
pharmaceutical formulation.
[0528] A "pharmaceutical composition" refers to a chemical or
biological composition suitable for administration to a mammal.
Such compositions may be specifically formulated for administration
via at least one of a number of routes, including but not limited
to buccal, epicutaneous, epidural, inhalation, intraarterial,
intracardial, intracerebroventricular, intradermal, intramuscular,
intranasal, intraocular, intraperitoneal, intraspinal, intrathecal,
intravenous, oral, parenteral, rectally via an enema or
suppository, subcutaneous, subdermal, sublingual, transdermal, and
transmucosal. In addition, administration can occur by means of
injection, powder, liquid, gel, drops, or other means of
administration. Further, a pharmaceutical composition comprising an
anti-IL-6 antibody described herein (e.g., ALD518) may be
administered subcutaneously.
[0529] In one embodiment of the subject technology, the anti-IL-6
antibodies described herein, or IL-6 binding fragments or variants
thereof, as well as combinations of said antibody fragments or
variants, may be optionally administered in combination with at
least one active agent. Such active agents include analgesic,
antipyretic, anti-inflammatory, antibiotic, antiviral, and
anti-cytokine agents. Active agents include agonists, antagonists,
and modulators of TNF-alpha, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13,
IL-18, IFN-alpha, IFN-gamma, BAFF, CXCL13, IP-10, VEGF, EPO, EGF,
HRG, Hepatocyte Growth Factor (HGF), Hepcidin, including antibodies
reactive against any of the foregoing, and antibodies reactive
against any of their receptors. Active agents also include
2-Arylpropionic acids, Aceclofenac, Acemetacin, Acetylsalicylic
acid (Aspirin), Alclofenac, Alminoprofen, Amoxiprin, Ampyrone,
Arylalkanoic acids, Azapropazone, Benorylate/Benorilate,
Benoxaprofen, Bromfenac, Carprofen, Celecoxib, Choline magnesium
salicylate, Clofezone, COX-2 inhibitors, Dexibuprofen,
Dexketoprofen, Diclofenac, Diflunisal, Droxicam, Ethenzamide,
Etodolac, Etoricoxib, Faislamine, fenamic acids, Fenbufen,
Fenoprofen, Flufenamic acid, Flunoxaprofen, Flurbiprofen,
Ibuprofen, Ibuproxam, Indometacin, Indoprofen, Kebuzone,
Ketoprofen, Ketorolac, Lornoxicam, Loxoprofen, Lumiracoxib,
Magnesium salicylate, Meclofenamic acid, Mefenamic acid, Meloxicam,
Metamizole, Methyl salicylate, Mofebutazone, Nabumetone, Naproxen,
N-Arylanthranilic acids, Oxametacin, Oxaprozin, Oxicams,
Oxyphenbutazone, Parecoxib, Phenazone, Phenylbutazone,
Phenylbutazone, Piroxicam, Pirprofen, profens, Proglumetacin,
Pyrazolidine derivatives, Rofecoxib, Salicyl salicylate,
Salicylamide, Salicylates, Sulfinpyrazone, Sulindac, Suprofen,
Tenoxicam, Tiaprofenic acid, Tolfenamic acid, Tolmetin, and
Valdecoxib. Antibiotics include Amikacin, Aminoglycosides,
Amoxicillin, Ampicillin, Ansamycins, Arsphenamine, Azithromycin,
Azlocillin, Aztreonam, Bacitracin, Carbacephem, Carbapenems,
Carbenicillin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin,
Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefepime,
Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime,
Cefprozil, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftobiprole,
Ceftriaxone, Cefuroxime, Cephalosporins, Chloramphenicol,
Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin,
Cloxacillin, Colistin, Co-trimoxazole, Dalfopristin,
Demeclocycline, Dicloxacillin, Dirithromycin, Doripenem,
Doxycycline, Enoxacin, Ertapenem, Erythromycin, Ethambutol,
Flucloxacillin, Fosfomycin, Furazolidone, Fusidic acid,
Gatifloxacin, Geldanamycin, Gentamicin, Glycopeptides, Herbimycin,
Imipenem, Isoniazid, Kanamycin, Levofloxacin, Lincomycin,
Linezolid, Lomefloxacin, Loracarbef, Macrolides, Mafenide,
Meropenem, Meticillin, Metronidazole, Mezlocillin, Minocycline,
Monobactams, Moxifloxacin, Mupirocin, Nafcillin, Neomycin,
Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin,
Oxytetracycline, Paromomycin, Penicillin, Penicillins,
Piperacillin, Platensimycin, Polymyxin B, Polypeptides, Prontosil,
Pyrazinamide, Quinolones, Quinupristin, Rifampicin, Rifampin,
Roxithromycin, Spectinomycin, Streptomycin, Sulfacetamide,
Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole,
Sulfonamides, Teicoplanin, Telithromycin, Tetracycline,
Tetracyclines, Ticarcillin, Tinidazole, Tobramycin, Trimethoprim,
Trimethoprim-Sulfamethoxazole, Troleandomycin, Trovafloxacin, and
Vancomycin. Active agents also include Aldosterone, Beclometasone,
Betamethasone, Corticosteroids, Cortisol, Cortisone acetate,
Deoxycorticosterone acetate, Dexamethasone, Fludrocortisone
acetate, Glucocorticoids, Hydrocortisone, Methylprednisolone,
Prednisolone, Prednisone, Steroids, and Triamcinolone. Antiviral
agents include but are not limited to abacavir, aciclovir,
acyclovir, adefovir, amantadine, amprenavir, an antiretroviral
fixed dose combination, an antiretroviral synergistic enhancer,
arbidol, atazanavir, atripla, brivudine, cidofovir, combivir,
darunavir, delavirdine, didanosine, docosanol, edoxudine,
efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors,
famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion
inhibitor, ganciclovir, gardasil, ibacitabine, idoxuridine,
imiquimod, imunovir, indinavir, inosine, integrase inhibitor,
interferon, interferon type I, interferon type II, interferon type
III, lamivudine, lopinavir, loviride, maraviroc, MK-0518,
moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues,
oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin,
protease inhibitor, reverse transcriptase inhibitor, ribavirin,
rimantadine, ritonavir, saquinavir, stavudine, tenofovir, tenofovir
disoproxil, tipranavir, trifluridine, trizivir, tromantadine,
truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine,
viramidine, zalcitabine, zanamivir, and zidovudine. Any suitable
combination of these active agents is also contemplated.
[0530] A "pharmaceutical excipient" or a "pharmaceutically
acceptable excipient" is a carrier, usually a liquid, in which an
active therapeutic agent is formulated. In one embodiment of the
subject technology, the active therapeutic agent is a humanized
antibody described herein, or at least one fragments or variants
thereof. The excipient generally does not provide any
pharmacological activity to the formulation, though it may provide
chemical and/or biological stability, and release characteristics.
Exemplary formulations can be found, for example, in Grennaro
(2005) [Ed.] Remington: The Science and Practice of Pharmacy
[21.sup.st Ed.]
[0531] As used herein "pharmaceutically acceptable carrier" or
"excipient" includes any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents that are physiologically compatible. In
one embodiment, the carrier is suitable for parenteral
administration. Alternatively, the carrier can be suitable for
intravenous, intraperitoneal, intramuscular, or sublingual
administration. Pharmaceutically acceptable carriers include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the pharmaceutical compositions of the
subject technology is contemplated. Supplementary active compounds
can also be incorporated into the compositions.
[0532] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The subject
technology contemplates that the pharmaceutical composition is
present in lyophilized form. The composition may be formulated as a
solution, microemulsion, liposome, or other ordered structure
suitable to high drug concentration. The carrier may be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol), and suitable mixtures thereof. The subject
technology further contemplates the inclusion of a stabilizer in
the pharmaceutical composition.
[0533] The antibodies and fragments thereof, of the present subject
technology thereof may be formulated into pharmaceutical
compositions of various dosage forms. For example, the antibody may
be ALD518, a humanized anti-interleukin-6 (anti-IL-6) monoclonal
immunoglobulin 1 (IgG1) antibody manufactured in the yeast Pichia
pastoris. ALD518 may be supplied as a pH 6.0 frozen injection in
single-use vials (80 mg or 160 mg) for intravenous administration.
Examplary non-active excipients include but are not limited to
histidine (e.g., 25 mM) and sorbitol (e.g., 250 mM). For example, a
160 mg formulation may comprise as non-active excipients, 25 mM
histidine, 250 mM sorbitol, and 0.015% polysorbate 80. To prepare
the pharmaceutical compositions of the subject technology, at least
one anti-IL-6 antibodies or binding fragments thereof, as the
active ingredient may be intimately mixed with appropriate carriers
and additives according to techniques well known to those skilled
in the art of pharmaceutical formulations. See Grennaro (2005)
[Ed.] Remington: The Science and Practice of Pharmacy [21.sup.st
Ed.] For example, the antibodies described herein may be formulated
in phosphate buffered saline pH 7.2 and supplied as a 5.0 mg/mL
clear colorless liquid solution.
[0534] Similarly, compositions for liquid preparations include
solutions, emulsions, dispersions, suspensions, syrups, and
elixirs, with suitable carriers and additives including but not
limited to water, alcohols, oils, glycols, preservatives, flavoring
agents, coloring agents, and suspending agents. Typical
preparations for parenteral administration comprise the active
ingredient with a carrier such as sterile water or parenterally
acceptable oil including but not limited to polyethylene glycol,
polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with
other additives for aiding solubility or preservation may also be
included. In the case of a solution, it may be lyophilized to a
powder and then reconstituted immediately prior to use. For
dispersions and suspensions, appropriate carriers and additives
include aqueous gums, celluloses, silicates, or oils.
[0535] For each of the recited embodiments, the anti-IL-6
antibodies or binding fragments thereof, may be administered by a
variety of dosage forms. Any biologically-acceptable dosage form
known to persons of ordinary skill in the art, and combinations
thereof, are contemplated. Examples of such dosage forms include,
without limitation, reconstitutable powders, elixirs, liquids,
solutions, suspensions, emulsions, powders, granules, particles,
microparticles, dispersible granules, cachets, inhalants, aerosol
inhalants, patches, particle inhalants, implants, depot implants,
injectables (including subcutaneous, intramuscular, intravenous,
and intradermal), infusions, and combinations thereof.
[0536] In many cases, it will be preferable to include isotonic
agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or
sodium chloride in the composition. Prolonged absorption of the
injectable compositions may be brought about by including in the
composition an agent which delays absorption, e.g., monostearate
salts and gelatin. Moreover, the compounds described herein may be
formulated in a time release formulation, e.g. in a composition
that includes a slow release polymer. The anti-IL-6 antibodies may
be prepared with carriers that will protect the compound against
rapid release, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers may be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters,
polylactic acid and polylactic, polyglycolic copolymers (PLG). Many
methods for the preparation of such formulations are known to those
skilled in the art.
[0537] In one embodiment of the subject technology that may be used
to intravenously administer antibodies of the subject technology,
including ALD518, for psoriatic arthritis indications, the
administration formulation comprises, or alternatively consists of,
about 10.5 mg/mL of antibody, 25 mM Histidine base, Phosphoric acid
q.s. to pH 6, and 250 mM sorbitol.
[0538] In another embodiment of the subject technology that may be
used to intravenously administer antibodies of the subject
technology, including ALD581, for psoriatic arthritis indications,
the administration formulation comprises, or alternatively consists
of, about 10.5 mg/mL of antibody, 12.5 mM Histidine base, 12.5 mM
Histidine HCl (or 25 mM Histidine base and Hydrochloric acid q.s.
to pH 6), 250 mM sorbitol, and 0.015% (w/w) Polysorbate 80.
[0539] In one embodiment of the subject technology that may be used
to subcutaneously administer antibodies of the subject technology,
including ALD518, for psoriatic arthritis indications, the
administration formulation comprises, or alternatively consists of,
about 50 or 100 mg/mL of antibody, about 5 mM Histidine base, about
5 mM Histidine HCl to make final pH 6, 250 mM sorbitol, and 0.015%
(w/w) Polysorbate 80. In another embodiment of the subject
technology that may be used to subcutaneously administer antibodies
of the subject technology, including Ab1, for psoriatic arthritis
indications, the administration formulation comprises, or
alternatively consists of, about 20 or 100 mg/mL of antibody, about
5 mM Histidine base, about 5 mM Histidine HCl to make final pH 6,
250 to 280 mM sorbitol (or sorbitol in combination with sucrose),
and 0.015% (w/w) Polysorbate 80, said formulation having a nitrogen
headspace in the shipping vials.
[0540] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The subject
technology contemplates that the pharmaceutical composition is
present in lyophilized form. The composition can be formulated as a
solution, microemulsion, liposome, or other ordered structure
suitable to high drug concentration. The carrier can be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol), and suitable mixtures thereof. The subject
technology further contemplates the inclusion of a stabilizer in
the pharmaceutical composition.
[0541] In many cases, it will be preferable to include isotonic
agents, for example, sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for
example, monostearate salts and gelatin. Moreover, the alkaline
polypeptide can be formulated in a time release formulation, for
example in a composition which includes a slow release polymer. The
active compounds can be prepared with carriers that will protect
the compound against rapid release, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, polylactic acid and polylactic,
polyglycolic copolymers (PLG). Many methods for the preparation of
such formulations are known to those skilled in the art.
[0542] For each of the recited embodiments, the compounds can be
administered by a variety of dosage forms. Any
biologically-acceptable dosage form known to persons of ordinary
skill in the art, and combinations thereof, are contemplated.
Examples of such dosage forms include, without limitation,
reconstitutable powders, elixirs, liquids, solutions, suspensions,
emulsions, powders, granules, particles, microparticles,
dispersible granules, cachets, inhalants, aerosol inhalants,
patches, particle inhalants, implants, depot implants, injectables
(including subcutaneous, intramuscular, intravenous, and
intradermal), infusions, and combinations thereof.
[0543] A person of skill in the art would be able to determine an
effective dosage and frequency of administration through routine
experimentation, for example guided by the disclosure herein and
the teachings in Goodman, et al. (2011) Goodman & Gilman's The
Pharmacological Basis of Therapeutics [12.sup.th Ed.]; Howland, et
al. (2005) Lippincott's Illustrated Reviews: Pharmacology [2.sup.nd
Ed.]; and Golan, (2008) Principles of Pharmacology: The
Pathophysiologic Basis of Drug Therapy [2.sup.nd Ed.] See, also,
Grennaro (2005) [Ed.] Remington: The Science and Practice of
Pharmacy [21.sup.st Ed.]
[0544] The above description of various illustrated embodiments of
the subject technology is not intended to be exhaustive or to limit
the subject technology to the precise form disclosed. While
specific embodiments of, and examples for, the subject technology
are described herein for illustrative purposes, various equivalent
modifications are possible within the scope of the subject
technology, as those skilled in the relevant art will recognize.
The teachings provided herein of the subject technology can be
applied to other purposes, other than the examples described
above.
[0545] These and other changes can be made to the subject
technology in light of the above detailed description. In general,
in the following claims, the terms used should not be construed to
limit the subject technology to the specific embodiments disclosed
in the specification and the claims. Accordingly, the subject
technology is not limited by the disclosure, but instead the scope
of the subject technology is to be determined entirely by the
following claims.
[0546] The subject technology may be practiced in ways other than
those particularly described in the foregoing description and
examples. Numerous modifications and variations of the subject
technology are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
[0547] Certain teachings related to methods for obtaining a clonal
population of antigen-specific B cells were disclosed in U.S.
Patent Application Publication No. 2007/0269868.
[0548] Certain teachings related to humanization of rabbit-derived
monoclonal antibodies and preferred sequence modifications to
maintain antigen binding affinity were disclosed in U.S. Patent
Application Publication No. 2009/0104187.
[0549] Certain teachings related to producing antibodies or
fragments thereof using mating competent yeast and corresponding
methods were disclosed in U.S. Patent Application Publication No.
2006/0270045.
[0550] Certain teachings related to anti-IL-6 antibodies, methods
of producing antibodies or fragments thereof using mating competent
yeast and corresponding methods were disclosed in U.S. Patent
Application Publication No. 2009/0104187.
[0551] Certain teachings related to anti-IL-6 antibodies and
methods of using those antibodies or fragments thereof to address
certain diseases and/or disorders were disclosed in U.S. Patent
Application Publication No. 2010/0150829.
[0552] Certain anti-IL-6 antibody polynucleotides and polypeptides
are disclosed in the sequence listing accompanying this patent
application filing, and the disclosure of said sequence listing is
herein incorporated by reference in its entirety.
[0553] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject technology, and are
not intended to limit the scope of what is regarded as the subject
technology. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations) but
some experimental errors and deviations should be allowed for.
Unless otherwise indicated, parts are parts by weight, molecular
weight is average molecular weight, temperature is in degrees
centigrade; and pressure is at or near atmospheric.
EXAMPLES
[0554] In the following examples, the term "Ab1" refers to an
antibody comprising the light chain sequence of SEQ ID NO: 702 and
the heavy chain sequence of SEQ ID NO: 704, except where the
context indicates otherwise. The laboratory designation "Ab1" also
encompasses an anti-IL-6 antibody also known as "clazakizumab,"
"ALD518" and "BMS-945429" comprising the light chain sequence of
SEQ ID NO: 19 and the heavy chain sequence of SEQ ID NO: 20.
Example 1
Production of Enriched Antigen-Specific B Cell Antibody Culture
[0555] Panels of antibodies are derived by immunizing traditional
antibody host animals to exploit the native immune response to a
target antigen of interest. Typically, the host used for
immunization is a rabbit or other host that produces antibodies
using a similar maturation process and provides for a population of
antigen-specific B cells producing antibodies of comparable
diversity, e.g., epitopic diversity. The initial antigen
immunization can be conducted using complete Freund's adjuvant
(CFA), and the subsequent boosts effected with incomplete adjuvant.
At about 50-60 days after immunization, preferably at day 55,
antibody titers are tested, and the Antibody Selection (ABS)
process is initiated if appropriate titers are established. The two
key criteria for ABS initiation are potent antigen recognition and
function-modifying activity in the polyclonal sera.
[0556] At the time positive antibody titers are established,
animals are sacrificed and B cell sources isolated. These sources
include: the spleen, lymph nodes, bone marrow, and peripheral blood
mononuclear cells (PBMCs). Single cell suspensions are generated,
and the cell suspensions are washed to make them compatible for low
temperature long term storage. The cells are then typically
frozen.
[0557] To initiate the antibody identification process, a small
fraction of the frozen cell suspensions are thawed, washed, and
placed in tissue culture media. These suspensions are then mixed
with a biotinylated form of the antigen that was used to generate
the animal immune response, and antigen-specific cells are
recovered using the Miltenyi magnetic bead cell selection
methodology. Specific enrichment is conducted using streptavidin
beads. The enriched population is recovered and progressed in the
next phase of specific B cell isolation.
Example 2
Production of Clonal, Antigen-Specific B Cell-Containing
Culture
[0558] Enriched B cells produced according to Example 1 are then
plated at varying cell densities per well in a 96 well microtiter
plate. Generally, this is at 50, 100, 250, or 500 cells per well
with 10 plates per group. The media is supplemented with 4%
activated rabbit T cell conditioned media along with 50K frozen
irradiated EL4B feeder cells. These cultures are left undisturbed
for 5-7 days at which time supernatant-containing secreted antibody
is collected and evaluated for target properties in a separate
assay setting. The remaining supernatant is left intact, and the
plate is frozen at -70.degree. C. Under these conditions, the
culture process typically results in wells containing a mixed cell
population that comprises a clonal population of antigen-specific B
cells, i.e., a single well will only contain a single monoclonal
antibody specific to the desired antigen.
Example 3
Screening of Antibody Supernatants for Monoclonal Antibody of
Desired Specificity and/or Functional Properties
[0559] Antibody-containing supernatants derived from the well
containing a clonal antigen-specific B cell population produced
according to Example 2 are initially screened for antigen
recognition using ELISA methods. This includes selective antigen
immobilization (e.g., biotinylated antigen capture by streptavidin
coated plate), non-specific antigen plate coating, or
alternatively, through an antigen build-up strategy (e.g.,
selective antigen capture followed by binding partner addition to
generate a heteromeric protein-antigen complex). Antigen-positive
well supernatants are then optionally tested in a
function-modifying assay that is strictly dependant on the ligand.
One such example is an in vitro protein-protein interaction assay
that recreates the natural interaction of the antigen ligand with
recombinant receptor protein. Alternatively, a cell-based response
that is ligand dependent and easily monitored (e.g., proliferation
response) is utilized. Supernatant that displays significant
antigen recognition and potency is deemed a positive well. Cells
derived from the original positive well are then transitioned to
the antibody recovery phase.
Example 4
Recovery of Single, Antibody-Producing B Cell of Desired Antigen
Specificity
[0560] Cells are isolated from a well that contains a clonal
population of antigen-specific B cells (produced according to
Example 2 or 3), which secrete a single antibody sequence. The
isolated cells are then assayed to isolate a single,
antibody-secreting cell. Dynal.RTM. (magnetic beads) streptavidin
beads are coated with biotinylated target antigen under buffered
medium to prepare antigen-containing microbeads compatible with
cell viability. Next antigen-loaded beads, antibody-producing cells
from the positive well, and a fluorescein isothiocyanate
(FITC)-labeled anti-host H&L IgG antibody (as noted, the host
can be any mammalian host, e.g., rabbit, mouse, rat) are incubated
together at 37.degree. C. This mixture is then re-pipetted in
aliquots onto a glass slide such that each aliquot has on average a
single, antibody-producing B-cell. The antigen-specific,
antibody-secreting cells are then detected through fluorescence
microscopy. Secreted antibody is locally concentrated onto the
adjacent beads due to the bound antigen and provides localization
information based on the strong fluorescent signal.
Antibody-secreting cells are identified via FITC detection of
antibody-antigen complexes formed adjacent to the secreting cell.
The single cell found in the center of this complex is then
recovered using a micromanipulator. The cell is snap-frozen in an
Eppendorf PCR tube for storage at -80.degree. C. until antibody
sequence recovery is initiated.
Example 5
Isolation of Antibody Sequences from Antigen-Specific B Cell
[0561] Antibody sequences are recovered using a combined RT-PCR
based method from a single isolated B-cell produced according to
Example 4 or an antigenic specific B cell isolated from the clonal
B cell population obtained according to Example 2. Primers are
designed to anneal in conserved and constant regions of the target
immunoglobulin genes (heavy and light), such as rabbit
immunoglobulin sequences, and a two-step nested PCR recovery step
is used to obtain the antibody sequence. Amplicons from each well
are analyzed for recovery and size integrity. The resulting
fragments are then digested with AluI to fingerprint the sequence
clonality. Identical sequences display a common fragmentation
pattern in their electrophoretic analysis. Significantly, this
common fragmentation pattern which proves cell clonality is
generally observed even in the wells originally plated up to 1000
cells/well. The original heavy and light chain amplicon fragments
are then restriction enzyme digested with HindIII and XhoI or
HindIII and BsiWI to prepare the respective pieces of DNA for
cloning. The resulting digestions are then ligated into an
expression vector and transformed into bacteria for plasmid
propagation and production. Colonies are selected for sequence
characterization.
Example 6
Recombinant Production of Monoclonal Antibody of Desired Antigen
Specificity and/or Functional Properties
[0562] Correct full-length antibody sequences for each well
containing a single monoclonal antibody is established and miniprep
DNA is prepared using Qiagen solid-phase methodology. This DNA is
then used to transfect mammalian cells to produce recombinant
full-length antibody. Crude antibody product is tested for antigen
recognition and functional properties to confirm the original
characteristics are found in the recombinant antibody protein.
Where appropriate, large-scale transient mammalian transfections
are completed, and antibody is purified through Protein A affinity
chromatography. K.sub.d is assessed using standard methods (e.g.,
Biacore.RTM.) as well as IC.sub.50 in a potency assay.
Example 7
Preparation of Antibodies that Bind Human IL-6
[0563] By using the antibody selection protocol described herein,
one can generate an extensive panel of antibodies. The antibodies
have high affinity towards IL-6 (single to double digit pM Kd) and
demonstrate potent antagonism of IL-6 in multiple cell-based
screening systems (T1165 and HepG2). Furthermore, the collection of
antibodies displays distinct modes of antagonism toward IL-6-driven
processes.
Immunization Strategy
[0564] Rabbits were immunized with huIL-6 (R&R). Immunization
consisted of a first subcutaneous (sc) injection of 100 .mu.g in
complete Freund's adjuvant (CFA) (Sigma) followed by two boosts,
two weeks apart, of 50 .mu.g each in incomplete Freund's adjuvant
(IFA) (Sigma). Animals were bled on day 55, and serum titers were
determined by ELISA (antigen recognition) and by non-radioactive
proliferation assay (Promega) using the T1165 cell line.
Antibody Selection Titer Assessment
[0565] Antigen recognition was determined by coating Immulon 4
plates (Thermo) with 1 .mu.g/mL of huIL-6 (50 .mu.L/well) in
phosphate buffered saline (PBS, Hyclone) overnight at 4.degree. C.
On the day of the assay, plates were washed 3 times with PBS/Tween
20 (PBST tablets, Calbiochem). Plates were then blocked with 200
.mu.L/well of 0.5% fish skin gelatin (FSG, Sigma) in PBS for 30
minutes at 37.degree. C. Blocking solution was removed, and plates
were blotted. Serum samples were made (bleeds and pre-bleeds) at a
starting dilution of 1:100 (all dilutions were made in FSG 50
.mu.L/well) followed by 1:10 dilutions across the plate (column 12
was left blank for background control). Plates were incubated for
30 minutes at 37.degree. C. Plates were washed 3 times with
PBS/Tween 20. Goat anti-rabbit Fc-HRP (Pierce) diluted 1:5000 was
added to all wells (50 .mu.L/well), and plates were incubated for
30 minutes at 37.degree. C. Plates were washed as described above.
50 .mu.L/well of TMB-Stable stop (Fitzgerald Industries) was added
to plates, and color was allowed to develop, generally for 3 to 5
minutes. The development reaction was stopped with 50 .mu.L/well
0.5 M HCl. Plates were read at 450 nm. Optical density (OD) versus
dilution was plotted using Graph Pad Prizm software, and titers
were determined.
Functional Titer Assessment
[0566] The functional activity of the samples was determined by a
T1165 proliferation assay. T1165 cells were routinely maintained in
modified RPMI medium (Hyclone) supplemented with HEPES, sodium
pyruvate, sodium bicarbonate, L-glutamine, high glucose,
penicillin/streptomycin, 10% heat inactivated fetal bovine serum
(FBS) (all supplements from Hyclone), 2-mercaptoethanol (Sigma),
and 10 ng/mL of huIL-6 (R&D). On the day of the assay, cell
viability was determined by trypan blue (Invitrogen), and cells
were seeded at a fixed density of 20,000 cells/well. Prior to
seeding, cells were washed twice in the medium described above
without human-IL-6 (by centrifuging at 13000 rpm for 5 minutes and
discarding the supernatant). After the last wash, cells were
resuspended in the same medium used for washing in a volume
equivalent to 50 .mu.L/well. Cells were set aside at room
temperature.
[0567] In a round-bottom, 96-well plate (Costar), serum samples
were added starting at 1:100, followed by a 1:10 dilution across
the plate (columns 2 to 10) at 30 .mu.L/well in replicates of 5
(rows B to F: dilution made in the medium described above with no
huIL-6). Column 11 was medium only for IL-6 control. 30 .mu.L/well
of huIL-6 at 4.times. concentration of the final EC50
(concentration previously determined) were added to all wells
(huIL-6 was diluted in the medium described above). Wells were
incubated for 1 hour at 37.degree. C. to allow antibody binding to
occur. After 1 hour, 50 .mu.L/well of antibody-antigen (Ab-Ag)
complex were transferred to a flat-bottom, 96-well plate (Costar)
following the plate map format laid out in the round-bottom plate.
On Row G, 50 .mu.L/well of medium were added to all wells (columns
2 to 11) for background control. 50 .mu.L/well of the cell
suspension set aside were added to all wells (columns 2 to 11, rows
B to G). On Columns 1 and 12 and on rows A and H, 200 .mu.L/well of
medium was added to prevent evaporation of test wells and to
minimize edge effect. Plates were incubated for 72 hours at
37.degree. C. in 4% CO.sub.2. At 72 hours, 20 .mu.L/well of
CellTiter96 (Promega) reagents was added to all test wells per
manufacturer protocol, and plates were incubated for 2 hours at
37.degree. C. At 2 h, plates were gently mixed on an orbital shaker
to disperse cells and to allow homogeneity in the test wells.
Plates were read at 490 nm wavelength. Optical density (OD) versus
dilution was plotted using Graph Pad Prizm software, and functional
titer was determined. A positive assay control plate was conducted
as described above using MAB2061 (R&D Systems) at a starting
concentration of 1 .mu.g/mL (final concentration) followed by 1:3
dilutions across the plate.
Tissue Harvesting
[0568] Once acceptable titers were established, the rabbit(s) were
sacrificed. Spleen, lymph nodes, and whole blood were harvested and
processed as follows:
[0569] Spleen and lymph nodes were processed into a single cell
suspension by disassociating the tissue and pushing through sterile
wire mesh at 70 .mu.m (Fisher) with a plunger of a 20 cc syringe.
Cells were collected in the modified RPMI medium described above
without huIL-6, but with low glucose. Cells were washed twice by
centrifugation. After the last wash, cell density was determined by
trypan blue. Cells were centrifuged at 1500 rpm for 10 minutes; the
supernatant was discarded. Cells were resuspended in the
appropriate volume of 10% dimethyl sulfoxide (DMSO, Sigma) in FBS
(Hyclone) and dispensed at 1 mL/vial. Vials were then stored at
-70.degree. C. for 24 h prior to being placed in a liquid nitrogen
(LN2) tank for long-term storage.
[0570] Peripheral blood mononuclear cells (PBMCs) were isolated by
mixing whole blood with equal parts of the low glucose medium
described above without FBS. 35 mL of the whole blood mixture was
carefully layered onto 8 mL of Lympholyte Rabbit (Cedarlane) into a
45 mL conical tube (Corning) and centrifuged 30 minutes at 2500 rpm
at room temperature without brakes. After centrifugation, the PBMC
layers were carefully removed using a glass Pasteur pipette (VWR),
combined, and placed into a clean 50 mL vial. Cells were washed
twice with the modified medium described above by centrifugation at
1500 rpm for 10 minutes at room temperature, and cell density was
determined by trypan blue staining After the last wash, cells were
resuspended in an appropriate volume of 10% DMSO/FBS medium and
frozen as described herein.
B Cell Culture
[0571] On the day of setting up B cell culture, PBMC, splenocyte,
or lymph node vials were thawed for use. Vials were removed from
LN2 tank and placed in a 37.degree. C. water bath until thawed.
Contents of vials were transferred into 15 mL conical centrifuge
tube (Corning) and 10 mL of modified RPMI described above was
slowly added to the tube. Cells were centrifuged for 5 minutes at
1.5K rpm, and the supernatant was discarded. Cells were resuspended
in 10 mL of fresh media. Cell density and viability was determined
by trypan blue. Cells were washed again and resuspended at 1E07
cells/80 .mu.L medium. Biotinylated huIL-6 (B huIL-6) was added to
the cell suspension at the final concentration of 3 .mu.g/mL and
incubated for 30 minutes at 4.degree. C. Unbound B huIL-6 was
removed with two 10 mL washes of phosphate-buffered (PBF): Ca/Mg
free PBS (Hyclone), 2 mM ethylenediamine tetraacetic acid (EDTA),
0.5% bovine serum albumin (BSA) (Sigma-biotin free). After the
second wash, cells were resuspended at 1E07 cells/80 .mu.L PBF. 20
.mu.L of MACS.RTM. streptavidin beads (Milteni)/10E7 cells were
added to the cell suspension. Cells were incubated at 4.degree. C.
for 15 minutes. Cells were washed once with 2 mL of PBF/10E7 cells.
After washing, the cells were resuspended at 1E08 cells/500 .mu.L
of PBF and set aside. A MACS.RTM. MS column (Milteni) was
pre-rinsed with 500 mL of PBF on a magnetic stand (Milteni). Cell
suspension was applied to the column through a pre-filter, and
unbound fraction was collected. The column was washed with 1.5 mL
of PBF buffer. The column was removed from the magnet stand and
placed onto a clean, sterile 5 mL Polypropylene Falcon tube. 1 mL
of PBF buffer was added to the top of the column, and positive
selected cells were collected. The yield and viability of positive
and negative cell fraction was determined by trypan blue staining
Positive selection yielded an average of 1% of the starting cell
concentration.
[0572] A pilot cell screen was established to provide information
on seeding levels for the culture. Three 10-plate groups (a total
of 30 plates) were seeded at 50, 100, and 200 enriched B
cells/well. In addition, each well contained 50K cells/well of
irradiated EL-4.B5 cells (5,000 Rads) and an appropriate level of T
cell supernatant (ranging from 1-5% depending on preparation) in
high glucose modified RPMI medium at a final volume of 250
.mu.L/well. Cultures were incubated for 5 to 7 days at 37.degree.
C. in 4% CO.sub.2.
Identification of Selective Antibody Secreting B Cells
[0573] Cultures were tested for antigen recognition and functional
activity between days 5 and 7.
Antigen Recognition Screening
[0574] The ELISA format used is as described above except 50 .mu.L
of supernatant from the B cell cultures (BCC) wells (all 30 plates)
was used as the source of the antibody. The conditioned medium was
transferred to antigen-coated plates. After positive wells were
identified, the supernatant was removed and transferred to a
96-well master plate(s). The original culture plates were then
frozen by removing all the supernatant except 40 .mu.L/well and
adding 60 .mu.L/well of 16% DMSO in FBS. Plates were wrapped in
paper towels to slow freezing and placed at -70.degree. C.
Functional Activity Screening
[0575] Master plates were then screened for functional activity in
the T1165 proliferation assay as described before, except row B was
media only for background control, row C was media+IL-6 for
positive proliferation control, and rows D-G and columns 2-11 were
the wells from the BCC (50 .mu.L/well, single points). 40 .mu.L of
IL-6 was added to all wells except the media row at 2.5 times the
EC50 concentration determined for the assay. After 1 h incubation,
the Ab/Ag complex was transferred to a tissue culture (TC) treated,
96-well, flat-bottom plate. 20 .mu.L of cell suspension in modified
RPMI medium without huIL-6 (T1165 at 20,000 cells/well) was added
to all wells (100 .mu.L final volume per well). Background was
subtracted, and observed OD values were transformed into % of
inhibition.
B Cell Recovery
[0576] Plates containing wells of interest were removed from
-70.degree. C., and the cells from each well were recovered with
5-200 .mu.L washes of medium/well. The washes were pooled in a 1.5
mL sterile centrifuge tube, and cells were pelleted for 2 minutes
at 1500 rpm.
[0577] The tube was inverted, the spin repeated, and the
supernatant carefully removed. Cells were resuspended in 100
.mu.L/tube of medium. 100 .mu.L biotinylated IL-6 coated
streptavidin M280 Dynabeads (Invitrogen) and 16 .mu.L of goat
anti-rabbit H&L IgG-FITC diluted 1:100 in medium was added to
the cell suspension.
[0578] 20 .mu.L of cell/beads/FITC suspension was removed, and 5
.mu.L droplets were prepared on a glass slide (Corning) previously
treated with Sigmacote (Sigma), 35 to 40 droplets/slide. An
impermeable barrier of paraffin oil (JT Baker) was added to
submerge the droplets, and the slide was incubated for 90 minutes
at 37.degree. C., 4% CO.sub.2 in the dark.
[0579] Specific B cells that produce antibody can be identified by
the fluorescent ring around them due to antibody secretion,
recognition of the bead-associated biotinylated antigen, and
subsequent detection by the fluorescent-IgG detection reagent. Once
a cell of interest was identified, the cell in the center of the
fluorescent ring was recovered via a micromanipulator (Eppendorf).
The single cell synthesizing and exporting the antibody was
transferred into a 250 .mu.L microcentrifuge tube and placed in dry
ice. After recovering all cells of interest, these were transferred
to -70.degree. C. for long-term storage.
Example 8
Yeast Cell Expression
[0580] Antibody genes: Genes were cloned and constructed that
directed the synthesis of a chimeric humanized rabbit monoclonal
antibody.
[0581] Expression vector: The vector contains the following
functional components: 1) a mutant ColE 1 origin of replication,
which facilitates the replication of the plasmid vector in cells of
the bacterium Escherichia coli; 2) a bacterial Sh ble gene, which
confers resistance to the antibiotic Zeocin.RTM. (phleomycin) and
serves as the selectable marker for transformations of both E. coli
and P. pastoris; 3) an expression cassette composed of the
glyceraldehyde dehydrogenase gene (GAP gene) promoter, fused to
sequences encoding the Saccharomyces cerevisiae alpha mating factor
pre pro secretion leader sequence, followed by sequences encoding a
P. pastoris transcriptional termination signal from the P. pastoris
alcohol oxidase I gene (AOX1). The Zeocin.RTM. (phleomycin)
resistance marker gene provides a means of enrichment for strains
that contain multiple integrated copies of an expression vector in
a strain by selecting for transformants that are resistant to
higher levels of Zeocin.RTM. (phleomycin).
[0582] P. pastoris strains: P. pastoris strains met1, lys3, ura3
and ade1 may be used. Although any two complementing sets of
auxotrophic strains could be used for the construction and
maintenance of diploid strains, these two strains are especially
suited for this method for two reasons. First, they grow more
slowly than diploid strains that are the result of their mating or
fusion. Thus, if a small number of haploid ade1 or ura3 cells
remain present in a culture or arise through meiosis or other
mechanism, the diploid strain should outgrow them in culture.
[0583] The second is that it is easy to monitor the sexual state of
these strains since diploid Ade+ colonies arising from their mating
are a normal white or cream color, whereas cells of any strains
that are haploid ade1 mutants will form a colony with a distinct
pink color. In addition, any strains that are haploid ura3 mutants
are resistant to the drug 5-fluoro-orotic acid (FOA) and can be
sensitively identified by plating samples of a culture on minimal
medium+uracil plates with FOA. On these plates, only
uracil-requiring ura3 mutant (presumably haploid) strains can grow
and form colonies. Thus, with haploid parent strains marked with
ade1 and ura3, one can readily monitor the sexual state of the
resulting antibody-producing diploid strains (haploid versus
diploid).
Methods
[0584] Construction of pGAPZ-Alpha Expression Vectors for
Transcription of Light and Heavy Chain Antibody Genes.
[0585] The humanized light and heavy chain fragments were cloned
into the pGAPZ expression vectors through a PCR directed process.
The recovered humanized constructs were subjected to amplification
under standard KOD polymerase (Novagen) kit conditions ((1)
94.degree. C., 2 minutes; (2) 94.degree. C., 30 seconds (3)
55.degree. C., 30 seconds; (4) 72.degree. C., 30 seconds-cycling
through steps 2-4 for 35 times; (5) 72.degree. C. 2 minutes)
employing the following primers (1) light chain forward
AGCGCTTATTCCGCTATCCAGATGACCCAGTC-the AfeI site is single underlined
(SEQ ID NO: 729). The end of the HSA signal sequence is double
underlined, followed by the sequence for the mature variable light
chain (not underlined); the reverse CGTACGTTTGATTTCCACCTTG (SEQ ID
NO: 730).
[0586] Variable light chain reverse primer. BsiWI site is
underlined, followed by the reverse complement for the 3' end of
the variable light chain. Upon restriction enzyme digest with AfeI
and BsiWI this enable insertion in-frame with the pGAPZ vector
using the human HAS leader sequence in frame with the human kapp
light chain constant region for export. (2) A similar strategy is
performed for the heavy chain. The forward primer employed is
AGCGCTTATTCCGAGGTGCAGCTGGTGGAGTC (SEQ ID NO: 731). The AfeI site is
single underlined. The end of the HSA signal sequence is double
underlined, followed by the sequence for the mature variable heavy
chain (not underlined). The reverse heavy chain primer is
CTCGAGACGGTGACGAGGGT (SEQ ID NO: 732). The XhoI site is underlined,
followed by the reverse complement for the 3' end of the variable
heavy chain. This enables cloning of the heavy chain in-frame with
IgG-.gamma.1 CH1-CH2-CH3 region previous inserted within pGAPZ
using a comparable directional cloning strategy.
[0587] Transformation of expression vectors into haploid ade1 ura3,
met1 and lys3 host strains of P. pastoris. All methods used for
transformation of haploid P. pastoris strains and genetic
manipulation of the P. pastoris sexual cycle are as described in
Higgins, D. R., and Cregg, J. M., Eds. 1998. Pichia Protocols.
Methods in Molecular Biology. Humana Press, Totowa, N.J.
[0588] Prior to transformation, each expression vector is
linearized within the GAP promoter sequences with AvrII to direct
the integration of the vectors into the GAP promoter locus of the
P. pastoris genome. Samples of each vector are then individually
transformed into electrocompetent cultures of the ade1, ura3, met1
and lys3 strains by electroporation and successful transformants
are selected on YPD Zeocin.RTM. (phleomycin) plates by their
resistance to this antibiotic. Resulting colonies are selected,
streaked for single colonies on YPD Zeocin.RTM. (phleomycin) plates
and then examined for the presence of the antibody gene insert by a
PCR assay on genomic DNA extracted from each strain for the proper
antibody gene insert and/or by the ability of each strain to
synthesize an antibody chain by a colony lift/immunoblot method.
Wung, et al. (1996) Biotechniques 21: 808-812. Haploid ade1, met1
and lys3 strains expressing one of the three heavy chain constructs
are collected for diploid constructions along with haploid ura3
strain expressing light chain gene. The haploid expressing heavy
chain genes are mated with the appropriate light chain haploid ura3
to generate diploid secreting protein.
[0589] Mating of haploid strains synthesizing a single antibody
chain and selection of diploid derivatives synthesizing tetrameric
functional antibodies. To mate P. pastoris haploid strains, each
ade1 (or met1 or lys3) heavy chain producing strain to be crossed
is streaked across a rich YPD plate and the ura3 light chain
producing strain is streaked across a second YPD plate (.about.10
streaks per plate). After one or two days incubation at 30.degree.
C., cells from one plate containing heavy chain strains and one
plate containing ura3 light chain strains are transferred to a
sterile velvet cloth on a replica-plating block in a cross hatched
pattern so that each heavy chain strain contain a patch of cells
mixed with each light chain strain. The cross-streaked replica
plated cells are then transferred to a mating plate and incubated
at 25.degree. C. to stimulate the initiation of mating between
strains. After two days, the cells on the mating plates are
transferred again to a sterile velvet on a replica-plating block
and then transferred to minimal medium plates. These plates are
incubated at 30.degree. C. for three days to allow for the
selective growth of colonies of prototrophic diploid strains.
Colonies that arose are picked and streaked onto a second minimal
medium plate to single colony isolate and purify each diploid
strain. The resulting diploid cell lines are then examined for
antibody production.
[0590] Putative diploid strains are tested to demonstrate that they
are diploid and contain both expression vectors for antibody
production. For diploidy, samples of a strain are spread on mating
plates to stimulate them to go through meiosis and form spores.
Haploid spore products are collected and tested for phenotype. If a
significant percentage of the resulting spore products are single
or double auxotrophs it may be concluded that the original strain
must have been diploid. Diploid strains are examined for the
presence of both antibody genes by extracting genomic DNA from each
and utilizing this DNA in PCR reactions specific for each gene.
[0591] Fusion of haploid strains synthesizing a single antibody
chain and selection of diploid derivatives synthesizing tetrameric
functional antibodies. As an alternative to the mating procedure
described above, individual cultures of single-chain antibody
producing haploid ade1 and ura3 strains are spheroplasted and their
resulting spheroplasts fused using polyethylene glycol/CaCl.sub.2.
The fused haploid strains are then embedded in agar containing 1 M
sorbitol and minimal medium to allow diploid strains to regenerate
their cell wall and grow into visible colonies. Resulting colonies
are picked from the agar, streaked onto a minimal medium plate, and
the plates are incubated for two days at 30.degree. C. to generate
colonies from single cells of diploid cell lines. The resulting
putative diploid cell lines are then examined for diploidy and
antibody production as described above.
[0592] Purification and analysis of antibodies. A diploid strain
for the production of full length antibody is derived through the
mating of met1 light chain and lys3 heavy chain using the methods
described above. Culture media from shake-flask or fermenter
cultures of diploid P. pastoris expression strains are collected
and examined for the presence of antibody protein via SDS-PAGE and
immunoblotting using antibodies directed against heavy and light
chains of human IgG, or specifically against the heavy chain of
IgG.
[0593] To purify the yeast secreted antibodies, clarified media
from antibody producing cultures are passed through a protein A
column and after washing with 20 mM sodium phosphate, pH 7.0,
binding buffer, protein A bound protein is eluted using 0.1 M
glycine HCl buffer, pH 3.0. Fractions containing the most total
protein are examined by Coomassie blue strained SDS-PAGE and
immunoblotting for antibody protein. Antibody is characterized
using the ELISA described above for IL-6 recognition.
[0594] Assay for Antibody Activity.
[0595] The recombinant yeast-derived humanized antibody is
evaluated for functional activity through the IL-6 driven T1165
cell proliferation assay and IL-6 stimulated HepG2 haptoglobin
assay described above.
Example 9
Acute Phase Response Neutralization by Intravenous Administration
of Anti-IL-6 Antibody Ab1
[0596] Human IL-6 can provoke an acute phase response in rats, and
one of the major acute phase proteins that is stimulated in the rat
is alpha-2 macroglobulin (A2M). A study was designed to assess the
dose of antibody Ab1 required to ablate the A2M response to a
single subcutaneous injection of 100 .mu.g of human IL-6 given one
hour after different doses (0.03, 0.1, 0.3, 1, and 3 mg/kg) of
antibody Ab1 administered intravenously (n=10 rats/dose level) or
polyclonal human IgG1 as the control (n=10 rats). Plasma was
recovered and the A2M was quantitated via a commercial sandwich
ELISA kit (ICL Inc., Newberg OR; cat. no.-E-25A2M). The endpoint
was the difference in the plasma concentration of A2M at the 24
hour time point (post-Ab1).
[0597] The ID50 for antibody Ab1 was 0.1 mg/kg with complete
suppression of the A2M response at the 0.3 mg/kg. This demonstrates
that the IL-6 may be neutralized in vivo by anti-IL-6 antibodies
described herein.
Example 10
Multi-Dose Pharmacokinetic Evaluation of Antibody Ab1 in Non-Human
Primates
[0598] Antibody Ab1 was dosed in a single bolus infusion to a
single male and single female cynomolgus monkey in phosphate
buffered saline. Plasma samples were removed at fixed time
intervals and the level of antibody Ab1 was quantitated through of
the use of an antigen capture ELISA assay. Biotinylated IL-6 (50
.mu.l of 3 ng/mL) was captured on Streptavidin coated 96 well
microtiter plates. The plates were washed and blocked with 0.5%
Fish skin gelatin. Appropriately diluted plasma samples were added
and incubated for 1 hour at room temperature. The supernatants
removed and an anti-hFc-HRP conjugated secondary antibody applied
and left at room temperature.
[0599] The plates were then aspirated and TMB added to visualize
the amount of antibody. The specific levels were then determined
through the use of a standard curve. A second dose of antibody Ab1
was administered at day 35 to the same two cynomolgus monkeys and
the experiment replicated using an identical sampling plan. The
resulting concentrations are then plot vs. time as show in FIG.
6.
[0600] This humanized full length aglycosylated antibody expressed
and purified Pichia pastoris displays comparable characteristics to
mammalian expressed protein. In addition, multiple doses of this
product display reproducible half-lives inferring that this
production platform does not generate products that display
enhanced immunogenicity.
Example 11
Octet Mechanistic Characterization of Antibody Proteins
[0601] IL-6 signaling is dependent upon interactions between IL-6
and two receptors, IL-6R1 (CD126) and gp130 (IL-6 signal
transducer). To determine the antibody mechanism of action,
mechanistic studies were performed using bio-layer interferometry
with an Octet QK instrument (ForteBio; Menlo Park, Calif.). Studies
were performed in two different configurations. In the first
orientation, biotinylated IL-6 (R&D systems part number
206-IL-001MG/CF, biotinylated using Pierce EZ-link
sulfo-NHS-LC-LC-biotin product number 21338 according to
manufacturer's protocols) was initially bound to a streptavidin
coated biosensor (ForteBio part number 18-5006). Binding is
monitored as an increase in signal.
[0602] The IL-6 bound to the sensor was then incubated either with
the antibody in question or diluent solution alone. The sensor was
then incubated with soluble IL-6R1 (R&D systems product number
227-SR-025/CF) molecule. If the IL-6R1 molecule failed to bind, the
antibody was deemed to block IL-6/IL-6R1 interactions. These
complexes were incubated with gp130 (R&D systems 228-GP-010/CF)
in the presence of IL-6R1 for stability purposes. If gp130 did not
bind, it was concluded that the antibody blocked gp130 interactions
with IL-6.
[0603] In the second orientation, the antibody was bound to a
biosensor coated with an anti-human IgG1 Fc-specific reagent
(ForteBio part number 18-5001). The IL-6 was bound to the
immobilized antibody and the sensor was incubated with IL-6R1. If
the IL-6R1 did not interact with the IL-6, then it was concluded
that the IL-6 binding antibody blocked IL-6/IL-6R1 interactions. In
those situations where antibody/IL-6/IL-6R1 was observed, the
complex was incubated with gp130 in the presence of IL-6R1. If
gp130 did not interact, then it was concluded that the antibody
blocked IL-6/130 interactions. All studies were performed in a 200
.mu.L final volume, at 30.degree. C. and 1000 rpm. For these
studies, all proteins were diluted using ForteBio's sample diluent
buffer (part number 18-5028). Results are presented in FIG. 7A-E
and TABLE 5.
TABLE-US-00005 TABLE 5 Anti-IL6 Antibodies binding to R1 or GP130
Antibody Blocks IL6 binding to R1 Blocks IL6 Binding to GP130 Ab1
Yes Yes Ab2 No Partial Ab3 No Yes Ab4 No Yes Ab6 Yes Yes Ab7 Yes
Yes Ab8 No Yes
Example 12
Peptide Mapping
[0604] In order to determine the epitope recognized by Ab1 on human
IL-6, the antibody was employed in a western-blot based assay. The
form of human IL-6 utilized in this example had a sequence of 183
amino acids in length. A 57-member library of overlapping 15 amino
acid peptides encompassing this sequence was commercially
synthesized and covalently bound to a PepSpots nitrocellulose
membrane (JPT Peptide technologies, Berlin, Germany). The sequences
of the overlapping 15 amino acid peptides are in SEQ ID NOs:
590-646. Blots were prepared and probed according to the
manufacturer's recommendations.
[0605] Briefly, blots were pre-wet in methanol, rinsed in PBS, and
blocked for over 2 hours in 10% non-fat milk in PBS/0.05% Tween
(Blocking Solution). The Ab1 antibody was used at 1 mg/mL final
dilution, and the HRP-conjugated Mouse Anti-Human-Kappa secondary
antibody (Southern BioTech #9220-05) was used at a 1:5000 dilution.
Antibody dilutions/incubations were performed in blocking solution.
Blots were developed using Amersham ECL advance reagents (GE#
RPN2135) and chemiluminescent signal documented using a CCD camera
(Alphalnnotec). The sequence of the form of human IL-6 utilized to
generate peptide library is set forth in SEQ ID NO: 1.
Example 13
Ab1 has High Affinity for IL-6
[0606] Surface plasmon resonance was used to measure association
rate (K.sub.a), dissociation rate (K.sub.d) and dissociation
constant (K.sub.D) for Ab1 to IL-6 from rat, mouse, dog, human, and
cynomolgus monkey at 25.degree. C. (TABLE 6). The dissociation
constant for human IL-6 was 4 pM, indicating very high affinity. As
expected, affinity generally decreased with phylogenetic distance
from human. The dissociation constants of Ab1 for IL-6 of
cynomolgus monkey, rat, and mouse were 31 pM, 1.4 nM, and 0.4 nM,
respectively. Ab1 affinity for dog IL-6 below the limit of
quantitation of the experiment.
[0607] The high affinity of Ab1 for mouse, rat, and cynomolgus
monkey IL-6 suggest that Ab1 may be used to inhibit IL-6 of these
species. This hypothesis was tested using a cell proliferation
assay. In brief, each species' IL-6 was used to stimulate
proliferation of T1165 cells, and the concentration at which Ab1
could inhibit 50% of proliferation (IC50) was measured Inhibition
was consistent with the measured dissociation constants (TABLE 7).
These results demonstrate that Ab1 can inhibit the native IL-6 of
these species, and suggest the use of these organisms for in vitro
or in vivo modeling of IL-6 inhibition by Ab1.
TABLE-US-00006 TABLE 6 Surface Plasmon Resonance: Averaged binding
constants determined at 25.degree. C. for Ab1 to IL-6. Species
(IL-6) K.sub.a (M.sup.-1s.sup.-1) K.sub.d (s.sup.-1) K.sub.p Rat
1.6e.sup.6 2.2e.sup.-3 1.4 nM Mouse 1.1e.sup.6 4.0e.sup.-4 0.4 nM
Dog Below LOQ.sup.a Below LOQ.sup.a Below LOQ.sup.a Human
1.6e.sup.5 5e.sup.-7 4 pM Cynomolgus 9.6e.sup.4 3e.sup.-6 31 pM
monkey .sup.aBelow Limit of Quantitation
TABLE-US-00007 TABLE 7 IC50 values for Ab1 against human,
cynomolgus monkey, mouse, rat and dog IL-6 in the T1165 assay. IL-6
Species IC50 (pM) Human 13 Cynomolgus monkey 12 Mouse 1840 Rat 2060
Dog No inhibition of cell proliferation
Example 14
Multi-Close Pharmacokinetic Evaluation of Antibody Ab1 in Healthy
Human Volunteers
[0608] Antibody Ab1 was dosed in a single bolus infusion in
histidine and sorbitol to healthy human volunteers. Dosages of 1
mg, 3 mg, 10 mg, 30 mg or 100 mg were administered to each
individual in dosage groups containing five to six individuals.
Plasma samples were removed at fixed time intervals for up to
twelve weeks. Human plasma was collected via venipuncture into a
vacuum collection tube containing EDTA. Plasma was separated and
used to assess the circulating levels of Ab1 using a monoclonal
antibody specific for Ab1, as follows. A 96 well microtiter plate
was coated overnight with the monoclonal antibody specific for Ab1
in 1.times.PBS overnight at 4.degree. C. The remaining steps were
conducted at room temperature. The wells were aspirated and
subsequently blocked using 0.5% Fish Skin Gelatin (FSG) (Sigma) in
1.times.PBS for 60 minutes. Human plasma samples were then added
and incubated for 60 minutes, then aspirated, then 50 .mu.L of 1
.mu.g/mL biotinylated IL-6 was then added to each well and
incubated for 60 minutes. The wells were aspirated, and 50 .mu.L
streptavidin-HRP (Pharmingen), diluted 1:5,000 in 0.5% FSG/PBS, was
added and incubated for 45 minutes. Development was conducted using
standard methods employing TMB for detection. Levels were then
determined via comparison to a standard curve prepared in a
comparable format.
[0609] Average plasma concentration of Ab1 for each dosage group
versus time is shown in TABLE 8. Mean AUC and C.sub.max increased
linearly with dosage. For dosages of 30 mg and above, the average
Ab1 half-life in each dosage group was between approximately 25 and
30 days.
Table 8. Summary of Ab1 Pharmacokinetics in Health Human
Volunteers
TABLE-US-00008 [0610] TABLE 8 Summary of Ab1 Pharmacokinetics in
Health Human Volunteers T.sub.1/2 AUC C.sub.max Dose of Ab1 (days)
(.mu.g h/mL) (.mu.g/mL) T.sub.max 1 mg 10.3 35 0.1 8 3 mg 11.6 229
0.7 4 10 mg 22.4 1473 4.0 4 30 mg 25.1 9076 19.7 4 100 mg 30.3
26128 48.0 12 300 mg 26.2 92891 188.0 12 640 mg 30.2 175684 306.0
12
Example 15
Pharmacokinetics of Ab1 in Patients with Advanced Cancer
[0611] Antibody Ab1 was dosed in a single bolus infusion in
phosphate buffered saline to five individuals with advanced cancer.
Each individual received a dosage of 80 mg (n=2) or 160 mg (n=3) of
Ab1. Plasma samples were drawn weekly, and the level of antibody
Ab1 was quantitated as in Example 16.
[0612] Average plasma concentration of Ab1 in these individuals as
a function of time is shown in FIG. 8. The average Ab1 half-life
was approximately 31 days.
Example 16
Half-Life of Ab1
[0613] Overall, the average half-life of Ab1 was approximately 31
days in humans (for dosages of 10 mg and above), and approximately
15-21 days in cynomolgus monkey. The Ab1 half-life in humans and
cynomolgus monkeys is unprecedented when compared with the
half-lives of other anti-IL-6 antibodies (TABLE 9). As described
above, Ab1 was derived from humanization of a rabbit antibody, and
is produced from Pichia pastoris in an aglycosylated form. These
characteristics results in an antibody with very low immunogenicity
in humans. Moreover, the lack of glycosylation prevents Ab1 from
interacting with the Fc receptor or complement. Without intent to
be limited by theory, it is believed that the unexpectedly long
half-life of Ab1 is at least partially attributable to the
humanization and/or the lack of glycosylation. The particular
sequence and/or structure of the antigen binding surfaces may also
contribute to Ab1's half-life.
TABLE-US-00009 TABLE 9 Elimination Half-life of Ab1 Cynomolgus
Monkey Human Dose of AB1 (days) (days) Ab1 15-21 ~31 Acemra
(Tocilizumab) 7 6 Remicade 5 8-9.5 Synagis 8.6 20 Erbitux 3-7 5
Zenapax 7 20 Avastin 10 20 Pertuzumab 10 18-22
Example 17
Ab1 Suppresses Serum CRP in Healthy Volunteers and in Patients with
Advanced Cancer
[0614] Introduction--
[0615] Serum CRP concentrations have been identified as a strong
prognostic indicator in patients with certain forms of cancer. For
example, Hashimoto et al. performed univariate and multivariate
analysis of preoperative serum CRP concentrations in patients with
hepatocellular carcinoma in order to identify factors affecting
survival and disease recurrence. Hashimoto, et al. (2005) Cancer
103(9): 1856-1864. Patients were classified into two groups, those
with serum CRP levels >1.0 mg/dL ("the CRP positive group") and
those with serum CRP levels <1.0 mg/dL ("the CRP negative
group"). The authors identified "a significant correlation between
preoperative serum CRP level and tumor size." Id. Furthermore, the
authors found that "[t]he overall survival and recurrence-free
survival rates in the CRP-positive group were significantly lower
compared with the rates in the CRP-negative group." Id. The authors
concluded that the preoperative CRP level of patients is an
independent and significant predictive indicator or poor prognosis
and early recurrence in patients with hepatocellular carcinoma.
[0616] Similar correlations have been identified by other
investigators. For example, Karakiewicz et al. determined that
serum CRP was an independent and informative predictor of renal
cell carcinoma-specific mortality. Karakiewicz, et al. (2007)
Cancer. 110(6):1241-1247. Accordingly, there remains a need in the
art for methods and/or treatments that reduce serum C-Reactive
Protein (CRP) concentrations in cancer patients, and particularly
those with advanced cancers.
[0617] Methods--
[0618] Healthy volunteers received a single 1-hour intravenous (IV)
infusion of either 100 mg (5 patients), 30 mg (5 patients), 10 mg
(6 patients), 3 mg (6 patients) or 1 mg (6 patients) of the Ab1
monoclonal antibody, while another 14 healthy volunteers received
intravenous placebo. Comparatively, 2 patients with advanced forms
of colorectal cancer received a single 1-hour intravenous (IV)
infusion of 80 mg of the Ab1 monoclonal antibody. No further
dosages of the Ab1 monoclonal antibody were administered to the
test population.
[0619] Patients were evaluated prior to administration of the
dosage, and thereafter on a weekly basis for at least 5 weeks post
dose. At the time of each evaluation, patients were screened for
serum CRP concentration.
[0620] Results--Healthy Volunteers
[0621] As noted above, serum CRP levels are a marker of
inflammation; accordingly, baseline CRP levels are typically low in
healthy individuals. The low baseline CRP levels can make a further
reduction in CRP levels difficult to detect. Nonetheless, a
substantial reduction in serum CRP concentrations was detectable in
healthy volunteers receiving all concentrations of the Ab1
monoclonal antibody, compared to controls (FIG. 9A). The reduction
in serum CRP levels was rapid, occurring within one week of
antibody administration, and prolonged, continuing at least through
the final measurement was taken (8 or 12 weeks from antibody
administration).
[0622] Results--Cancer Patients
[0623] Five advanced cancer patients (colorectal cancer,
cholangiocarcinoma, or NSCLC) having elevated serum CRP levels were
dosed with 80 mg or 160 mg of Ab1. Serum CRP levels were greatly
reduced in these patients (FIG. 9B). The reduction in serum CRP
levels was rapid, with 90% of the decrease occurring within one
week of Ab1 administration, and prolonged, continuing at least
until the final measurement was taken (up to twelve weeks). In two
representative individuals, the CRP levels were lowered to below
the normal reference range (less than 5-6 mg/1) within one week.
Thus, administration of Ab1 to patients can cause a rapid and
sustained suppression of serum CRP levels.
Example 18
Ab1 Suppresses Serum CRP in Patients with Advanced Cancer
[0624] Introduction--
[0625] Serum CRP concentrations have been identified as a strong
prognostic indicator in patients with certain forms of cancer. For
example, Hashimoto et al. performed univariate and multivariate
analysis of preoperative serum CRP concentrations in patients with
hepatocellular carcinoma in order to identify factors affecting
survival and disease recurrence. Hashimoto, et al. (2005) Cancer
103(9): 1856-1864. Patients were classified into two groups, those
with serum CRP levels >1.0 mg/dL ("the CRP positive group") and
those with serum CRP levels <1.0 mg/dL ("the CRP negative
group"). The authors identified "a significant correlation between
preoperative serum CRP level and tumor size." Id. Furthermore, the
authors found that "[t]he overall survival and recurrence-free
survival rates in the CRP-positive group were significantly lower
compared with the rates in the CRP-negative group." Id. The authors
concluded that the preoperative CRP level of patients is an
independent and significant predictive indicator of poor prognosis
and early recurrence in patients with hepatocellular carcinoma.
[0626] Similar correlations have been identified by other
investigators. For example, Karakiewicz et al. determined that
serum CRP was an independent and informative predictor of renal
cell carcinoma-specific mortality. Karakiewicz, et al. (2007)
Cancer 110(6):1241-1247. Accordingly, there remains a need in the
art for methods and/or treatments that reduce serum C-Reactive
Protein (CRP) concentrations in cancer patients, and particularly
those with advanced cancers.
[0627] Methods--
[0628] One-hundred twenty-four patients with non-small cell lung
cancer (NSCLC) were divided into 4 treatment groups. Patients in
one group received one 1-hour intravenous (IV) infusion of either
placebo (n=31), 80 mg (n=29), 160 mg (n=32), or 320 mg (n=32) of
the Ab1 monoclonal antibody every 8 weeks over a 24 week duration
for a total of 3 doses. CRP concentration was quantitated by a
C-reactive protein particle-enhanced immunoturbidimetric assay
using latex-attached anti-CRP antibodies (i.e. Roche CRP
Tinaquant.RTM.). Briefly, about 1.0 mL of patient sample serum was
collected and stored in a plastic collection tube. Sample was
placed into appropriate buffer, and anti-CRP antibody coupled to
latex microparticles was added to the sample to start the reaction.
These anti-CRP antibodies with conjugated latex microparticles
react with antigen in the sample to form an antigen/antibody
complex. Following agglutination, this was measured
turbidimetrically using a Roche/Hitachi Modular P analyzer.
[0629] Patients were evaluated prior to administration of the
dosage, and thereafter at weeks 2, 4, 8, and 12. At the time of
each evaluation, patients were screened for serum CRP
concentration.
[0630] Results--
[0631] The averaged data for each dosage concentrations (placebo,
80 mg, 160 mg, and 320 mg) of the Ab1 monoclonal antibody are
plotted in FIG. 10. All dosage levels of Ab1 antibody demonstrated
an immediate drop in CRP concentrations relative to placebo over
the period of 12 weeks. CRP levels displayed breakthrough at 8
weeks post-dosing. The CRP levels fell below 5 mg/L by week 12.
Median values of CRP demonstrated rapid and sustained decreases for
all dosage concentrations relative to placebo (FIG. 11). Thus,
administration of Ab1 to advanced cancer patients can cause a rapid
and sustained suppression of serum CRP levels.
Example 19
Ab1 Suppresses Serum CRP in Patients with Advanced Cancers
[0632] Introduction--
[0633] Serum CRP concentrations have been identified as a strong
prognostic indicator in patients with certain forms of cancer. For
example, Hashimoto et al. performed univariate and multivariate
analysis of preoperative serum CRP concentrations in patients with
hepatocellular carcinoma in order to identify factors affecting
survival and disease recurrence. Hashimoto, et al. (2005) Cancer
103(9): 1856-1864. Patients were classified into two groups, those
with serum CRP levels >1.0 mg/dL ("the CRP positive group") and
those with serum CRP levels <1.0 mg/dL ("the CRP negative
group"). The authors identified "a significant correlation between
preoperative serum CRP level and tumor size." Id. Furthermore, the
authors found that "[t]he overall survival and recurrence-free
survival rates in the CRP-positive group were significantly lower
compared with the rates in the CRP-negative group." Id. The authors
concluded that the preoperative CRP level of patients is an
independent and significant predictive indicator of poor prognosis
and early recurrence in patients with hepatocellular carcinoma.
[0634] Similar correlations have been identified by other
investigators. For example, Karakiewicz et al. determined that
serum CRP was an independent and informative predictor of renal
cell carcinoma-specific mortality. Karakiewicz, et al. (2007)
Cancer 110(6): 1241-1247. Accordingly, there remains a need in the
art for methods and/or treatments that reduce serum C-Reactive
Protein (CRP) concentrations in cancer patients, and particularly
those with advanced cancers.
[0635] Methods--
[0636] Eight patients with various forms of advanced cancer
(colorectal (3), NSCLC (1), cholangio (1), and mesothelioma (2))
received a single 1-hour intravenous infusion of either 80 mg (2
patients), 160 mg (3 patients) or 320 mg (3 patients) of the Ab1
monoclonal antibody. No further dosages of the Ab1 monoclonal
antibody were administered to the test population.
[0637] Patients were evaluated prior to administration of the
dosage and thereafter on a weekly basis for at least 8 weeks post
dose. At the time of each evaluation, patients were screened for
serum CRP concentration. CRP concentration was quantitated by a
C-reactive protein particle-enhanced immunoturbidimetric assay
using latex-attached anti-CRP antibodies (i.e. Roche CRP
Tinaquant.RTM.). Briefly, about 1.0 mL of patient sample serum was
collected and stored in a plastic collection tube. Sample was
placed into appropriate buffer, and anti-CRP antibody coupled to
latex microparticles was added to the sample to start the reaction.
These anti-CRP antibodies with conjugated latex microparticles
react with antigen in the sample to form an antigen/antibody
complex. Following agglutination, this was measured
turbidimetrically using a Roche/Hitachi Modular P analyzer.
[0638] Results--
[0639] Serum CRP levels were greatly reduced in all patients
studied (FIG. 12). The reduction in serum CRP levels was rapid,
with approximately 90% of the decrease occurring within one week of
Ab1 administration, and prolonged diminished levels continued at
least until the final measurement was taken (up to twelve weeks).
In all cases except one patient with colorectal cancer, CRP levels
fell to at or below the normal reference range (less than 5-6 mg/L)
within one week. The colorectal cancer patient achieved similar
normal levels by week 4 of the study. Thus, administration of Ab1
to advanced cancer patients can cause a rapid and sustained
suppression of serum CRP levels.
Example 20
Safety, Pharmacokinetics (PK), and Pharmacodynamics (PD) of Ab1 in
Human Subjects
[0640] Background--
[0641] A humanized antibody derived from Ab1 (humanized Ab1 or
ALD518) containing the variable heavy and light sequences in SEQ ID
NO: 19 and 20 was administered to rheumatoid arthritis patients.
This antibody is a humanized, asialated, IgG1 monoclonal antibody
against IL-6 which has been shown to have a half-life (t1/2) of
approximately 30 days in humans. In studies in patients with RA,
intravenous (IV) with this antibody (humanized Ab1) has
demonstrated: efficacy over 16 weeks with rapid American College of
Rheumatology (ACR) responses; Complete and durable suppression of
C-reactive protein (CRP); Good tolerability, and a safety profile
consistent with the biology of IL-6 blockade. This humanized
antibody binds to IL-6 with high affinity, preventing interaction
and signalling mediated via IL-6R. Rapid and significant treatment
responses have been demonstrated with intravenous (IV)
administration of humanized Ab1 in patients with RA. In this
example we study the safety, pharmacokinetics and pharmacodynamics
of subcutaneous (SC) administration of humanized Ab1 in healthy
subjects.
[0642] The objective of this study was to assess the safety,
pharmacokinetics (PK) and pharmacodynamics (PD) of a single SC
injection of this humanized antibody in healthy male subjects.
[0643] Methods--
[0644] In this Phase I, double-blind, placebo-controlled study, 27
subjects were randomized 2:1 to receive a single dose of humanized
Ab1 or placebo in the following groups: humanized Ab1 50 mg SC,
humanized Ab1100 mg SC or humanized Ab1100 mg IV (n=6 active and
n=3 placebo per group). The primary objective was to assess safety
of SC humanized Ab1 versus placebo over 12 weeks. Plasma
concentrations of humanized Ab1 and serum concentrations of
C-reactive protein (CRP) were assessed as secondary objectives.
Assessments were performed daily in Week 1 and then on Day 10,
Weeks 2, 4, 6 and 8, and then monthly to Week 12. The study was
unblinded at Week 12, and humanized Ab1 subjects were monitored to
Week 24.
[0645] Study Design and Population--
[0646] The study included 27 healthy male subjects (aged 18-65
years). Subjects were dosed in three treatment groups of nine
subjects each, randomized 2:1 to receive a single dose of humanized
Ab1 or placebo on Day 1. Humanized Ab1 treatments per group were:
humanized Ab1 IV 100 mg infusion over 60 minutes; humanized Ab1 SC
50 mg injection (1 mL); or humanized Ab1100 mg injection (1 mL).
The study was unblinded at Week 12, after which placebo subjects
discontinued the trial and ALD518 subjects were monitored to Week
24.
[0647] Safety and Immunogenicity Assessments--
[0648] The primary objective of the study was to assess the safety
of SC humanized Ab1 compared with placebo over 12 weeks. Safety was
monitored over 12 weeks for all subjects. The study was unblinded
at Week 12, and Humanized AB1 subjects were monitored to Week 24.
Laboratory safety tests were performed pre-dose at screening and
Day -1, and post dose on Days 2 and 7, Weeks 2, 4, 6, 8 and 12 for
all subjects, and Weeks 16, 20 and 24 post-dose for those
randomized to Humanized Ab1. Anti-Humanized AB1 antibodies were
measured by enzyme-linked immunosorbent assay (ELISA). Blood
samples were collected at Day 1 (pre-dose) and Week 12 post-dose
for all subjects, and Week 24 post-dose for those randomized to
Humanized Ab1.
[0649] Pharmacokinetic and Pharmacodynamic Assessments--
[0650] Plasma Humanized AB1 and serum CRP concentrations were
assessed by ELISA. For all subjects, samples were collected at
screening, pre-dose on Day 1, and post-dose on Days 2 and 7 and
Weeks 2, 4, 6, 8 and 12. For subjects randomized to Humanized AB1,
further samples were collected at Weeks 16, 20 and 24
post-dose.
[0651] Statistical Analysis--
[0652] All subjects who received a dose of Humanized AB1 or placebo
were included in the safety analysis. All subjects who received a
dose of Humanized AB1 or placebo were included in PD and
immunogenicity analyses. All subjects who received a dose of
Humanized AB1 were included in PK analyses (n=18). All PK samples
for placebo subjects were confirmed as below quantification.
Descriptive statistics were generated for baseline demographics,
safety data, plasma Humanized AB1 parameters and serum CRP
concentrations. Wilcoxon Rank Sum test was used to compare CRP
concentrations for Humanized AB1 treatments versus placebo.
[0653] Results--Summary
[0654] Over 24 weeks, there were no deaths or serious AEs, and no
withdrawals due to AEs. Nearly all subjects (89%) experienced AEs,
which were mild or moderate except one event of severe
gastroenteritis in the Humanized ab1 SC 50 mg group. Injection site
reactions occurred in 5/12 Humanized Ab1 SC subjects, 1/6 placebo
SC subjects and 1/3 placebo IV subjects (none were reported in
Humanized Ab1 IV subjects). These were mild except one case of
moderate erythema and pruritis in the Humanized Ab1 100 mg SC
group. Increases in direct bilirubin and neutrophil counts below
the limit of normal were more common in subjects receiving
Humanized Ab1 than placebo; all were CTC Grade 1 or 2. The
half-life of Humanized Ab1 was similar across all groups (mean
range: 30.7-33.6 days). The median Tmax of Humanized Ab1 was longer
after SC (.about.1 week) than after IV administration (.about.end
of infusion). The PK of SC Humanized Ab1 was dose-proportional in
terms of AUC and Cmax at doses of 50 mg and 100 mg. Based on
AUCO-.infin. (day*.mu.g/mL) of 237, 452 and 764 for the Humanized
Ab1 50 mg SC, 100 mg SC and 100 mg IV groups, respectively, the
bioavailability of Humanized Ab1 was .about.60% for the SC versus
IV groups. Subjects receiving Humanized Ab1 experienced rapid and
sustained reductions in serum CRP (FIG. 13), similar results were
seen when the antibody was administered either intravenous or
subcutaneously (FIG. 14).
[0655] Subject Disposition and Baseline Demographics--
[0656] A total of 27 subjects were enrolled and completed the study
(n=18 Humanized Ab1 and n=9 placebo). No subjects were withdrawn
for any reason. All subjects were male; 23/27 subjects were
Caucasian and 4/27 were Asian. Mean age was 29 (range 20-59) and
was similar across the groups. Mean height and weight were also
generally comparable across groups, although the IV placebo group
were slightly lighter.
[0657] Safety and immunogenicity to Week 12 for Humanized AB1 and
placebo--A summary of safety is presented in TABLE 10. For the SC
Humanized AB1 groups, a total of 11/12 (91%) patients experienced
an adverse event (AE) compared with: 6/6 (100%) for the IV
Humanized AB1 group; 4/6 (66.6%) for the SC placebo group; and 3/3
(100%) for the IV placebo group.
TABLE-US-00010 TABLE 10 Adverse Events Up to Week 12 Week 12-Week
24* MedRA SC 50 mg SC 100 mg IV 100 mg Placebo SC Placebo IV SC 100
mg SC 100 mg IV 100 mg Preferred Term n = 6 n = 6 n = 6 n = 6 n = 6
n = 6 n = 6 n = 6 Subjects with 6 5 6 4 3 3 5 5 an AE AE severity
Mild 2 2 5 1 2 3 5 7 Moderate 3 3 1 3 1 1 1 0 Severe 1 0 0 0 0 0 0
0 Discontinuations 0 0 0 0 0 0 0 0 Due to AEs Deaths 0 0 0 0 0 0 0
0 AEs reported in .gtoreq.2 subjects in any group Injection site 1
2 0 0 0 0 0 0 erythema Injection site 1 2 0 0 1 0 0 0 pruritis
Gastroenteritis 1 0 2 0 0 0 0 0 URTI 4 4 4 2 2 0 1 2 Skin
laceration 2 1 2 0 0 0 0 0 Myalgia 0 0 0 2 0 0 0 0 Headache 5 2 1 1
0 0 1 1 Nasal congestion 0 0 2 0 0 0 0 0 *Patients randomized to
placebo (IV or SC) discontinued at Week 12 and are not included in
Week 24 analyses; AE = adverse event; SC = subcutaneous; IV =
intravenous; URTI = upper respiratory tract infection.
[0658] Across groups: No deaths or serious AEs were reported and
there were no withdrawals due to AEs. Most AEs were mild or
moderate in intensity. One case of gastroenteritis in a SC
Humanized AB1 50 mg subject was considered severe, but not serious,
and not related to study medication. No anti-Humanized AB1
antibodies were detected in any subject during this period.
[0659] Injection Site Reactions--
[0660] Injection site reactions were reported in 26% (7/27) of
subjects, and all occurred prior to Week 12 (TABLE 11). Injection
site reactions occurred in 5/12 SC Humanized AB1 subjects and 1/6
SC placebo subjects. In the IV groups, 0/6 Humanized AB1 subjects
and 1/3 placebo subjects experienced injection site reactions. All
injection site reactions were mild except in one SC Humanized AB 1
100 mg subject with moderate injection site erythema and pruritis.
No injection site reactions occurred after Week 12 in any of the
Humanized AB1 groups. Infusion site reactions were reported in 0/6
subjects receiving IV Humanized AB1 and 1/3 IV placebo subjects
(infusion site pruritis)
TABLE-US-00011 TABLE 11 Ab1 Injection Site Reactions to Week 12*
Placebo Placebo 50 mg 100 mg 100 mg SC IV n = 6 n = 6 n = 6 n = 6 n
= 3 Total subjects with 2 3 0 1 1 injection site reaction Injection
site erythema 1 2 0 0 0 Injection site pain 1 1 0 1 0 Injection
site pruritis 1 2 0 0 1 Injection site rash 1 0 0 0 0 *All
injection site reactions were reported in the first 12 weeks of the
study. SC = subcutaneous; IV = intravenous
[0661] Clinical Laboratory Evaluations--
[0662] TABLE 12 shows incidences of increased alanine
aminotransferase (ALT) and aspartate aminotransferase (AST) and
bilirubin levels across the Humanized AB1 and placebo groups. All
ALT and AST levels were Grade 1 by the Common Terminology Criteria
for Adverse Events (CTCAE), and no levels were .gtoreq.3 times the
upper limit of normal (ULN). All increases in total and direct
bilirubin were CTCAE Grade 1 or 2 and no subject met criteria for
drug-induced liver damage. Only one subject (SC Humanized AB1 100
mg group) had total bilirubin out of range (26 .mu.mol/L, range
0-24 .mu.mol/L), at Week 24.
TABLE-US-00012 TABLE 12 Clinical Laboratory Evaluations Over 24
Weeks (Ab1) SC 50 mg SC 100 mg IV 100 mg Placebo* n = 6 n = 6 n = 6
n = 9 Elevated ALT 0 1 3 2 Elevated AST 0 1 1 1 Elevated total
bilirubin 0 1 1 0 Elevated direct 2 4 5 2 bililrubin Low neutrophil
count.sup..dagger. 4 1 2 3 Low platelet count.sup..dagger. 2 0 0 1
*SC and IV groups combined up to Week 12 only, after which
placebo-treated patients discontinued; .sup..dagger.Below the lower
limit of normal; SC = subcutaneous; IV = intravenous; ALT = alanine
aminotransferase; AST = aspartate aminotransferase
[0663] Sporadic decreases in neutrophil and platelet counts were
also observed in the Humanized AB1 and placebo groups. Neutrophil
counts below the lower limit of normal were more common in subjects
receiving Humanized AB 1 than placebo but all decreases were CTCAE
Grade 1 or 2. Only one subject (SC Humanized AB1 50 mg group) had
consistent mild neutropenia to Week 24 (1.6.times.10.sup.9/L at
Week 24). Reductions in platelet counts were all CTCAE Grade 1
(lowest level 134.times.10.sup.9/L) and no subject had a low
platelet count past Week 8.
[0664] Pharmacokinetics--
[0665] Bioavailability of Humanized AB1 was 60% for SC Humanized
AB1 50 and 100 mg versus IV Humanized AB1 100 mg groups based on
the mean AUC.sub.0-.infin.. (TABLE 13). The half-life of Humanized
AB1 was similar across all groups (mean range: 30.7-33.6 days)
(Table G). Peak plasma concentration (C.sub.max) of SC Humanized
AB1 was reduced as compared to IV (FIG. 15). Median time to maximum
plasma concentration (T.sub.max) of Humanized Ab1 was longer after
SC Humanized AB1 (at approximately one week) than after IV
Humanized Ab1 administration (at approximately the end of
infusion).
TABLE-US-00013 TABLE 13 Ab1 Plasma Pharmacokinetic Parameters to
Week 24 SC 50 mg SC 100 mg IV 100 mg n = 6 n = 6 n = 6 C.sub.max
(.mu.g/mL) (CV)* 5.57 (24%) 9.19 (34%) 33.6 (30%) T.sub.max (days)
(min, max).sup..dagger. 6 (6, 14) 5.5 (2, 28) 0.17 (0, 17, 0.34)
AUC.sub.5-24 (day .mu.g/mL) (CV)* 218 (34%) 435 (19%) 732 (22%)
AUC.sub.8-.gamma. (day .mu.g/mL) (CV)* 224 (39%) 444 (20%) 746
(22%) t.sub.1/2 (days .+-. SD).sup..dagger-dbl. 33.6 .+-. 21.7 31.1
.+-. 9.0 30.7 .+-. 5.9 CL (mL/day) (CV)* 223 (32%) 225 (21%) 134
(27%) *Data are geometric mean (coefficient of variation %, CV %).
.sup..dagger.Data are median (minimum, maximum).
.sup..dagger-dbl.Data are mean (.+-.SD). CV = coefficient of
variation; C.sub.max = maximum plasma concentration; AUC = area
under curve; SD = standard deviation; CL = apparent total body
clearance for IV and apparent total body clearance divided by
bioavailability for SC; IV = intravenous; SC = subcutaneous;
T.sub.max = time to maximum plasma concentration; t.sub.1/2 =
terminal plasma half-life
[0666] Pharmacodynamics--
[0667] CRP levels were reduced in all subjects who received
Humanized AB1 irrespective of dose or administration route. From
Weeks 4 to 12, CRP levels were significantly lower in subjects who
received Humanized Ab1 compared with placebo (unadjusted p-value
<0.05). A high correlation between the IgG produced and antigen
specificity for an exemplary IL-6 protocol was observed with 9 of
11 wells showed specific IgG correlation with antigen recognition.
In Humanized AB1 subjects, CRP levels were lowered to <20% of
pre-dose levels in: 72% (13/18) of subjects at Week 1; 73% (11/15)
of subjects at Week 12; and 56% (10/18) of subjects at Week 24.
[0668] Conclusions--
[0669] In this Phase I study, the anti-IL-6 antibody Humanized Ab1
was generally well tolerated when administered in a single SC dose
in healthy male subjects. Injection site reactions were generally
mild. No anti-Humanized Ab1 antibodies were detected. Changes in
liver enzymes, neutrophil and platelet counts were reversible. The
bioavailability of SC Humanized AB1 was approximately 60% of that
observed with IV Humanized Ab1. The half-life of Humanized AB1 was
approximately 30 days, irrespective of route of administration.
These data concur with previous data using IV Humanized Ab12.
Subcutaneous Humanized AB1 led to rapid and large reductions in
serum CRP. Reductions in CRP observed during the first 12 weeks of
the study were sustained over 24 weeks of assessment. These
preliminary data support the continued development and evaluation
of subcutaneous Humanized Ab1 for the treatment of patients with
psoriatic arthritis.
[0670] In summary, in this Phase I study, the anti-IL-6 antibody
Humanized Ab1 was well tolerated when administered in a single SC
dose; injection site reactions were generally mild. The
bioavailability of SC Humanized Ab1 was .about.60% of IV Humanized
Ab1, and the half-life was .about.30 days. Rapid and significant
reductions in CRP were observed, which were sustained over 24 weeks
of assessment.
Example 21
Randomized, Double-Blind, Placebo-Controlled, Dose Ranging,
Multi-Center (Phase IIIB) Study to Evaluate the Efficacy and Safety
of BMS-945429 ("Clazakizumab") Subcutaneous Injection in Adults
with Active Psoriatic Arthritis
[0671] Background--
[0672] Psoriatic arthritis (PsA), a seronegative
spondyloarthropathy is a complex disease involving peripheral and
axial joints, periarticular structures (e.g., enthesitis,
inflammation of other soft tissues, dactylitis) as well as the skin
and nails. Without appropriate management, the number of joints
affected by PsA and the severity of joint damage increase over
time, which can lead to marked restrictions of the daily activities
and to substantially compromised quality of life. Evidence has
shown that accelerated atherosclerosis, obesity, metabolic syndrome
and cardiovascular disease are associated with active PsA. Other
co-morbidities such as pulmonary fibrosis, uveitis, and, less
commonly, aortic and aortic valve inflammation also contribute to
complexity of PsA.
[0673] Unlike RA, effective treatment options are limited for PsA.
Saad et al. (2008) J Rheumatol. 35:883-90; Kavanaugh et al. (2006)
J Rheumatol. 33:1417-21; Lee Gavin (2010) The Hong Kong Medical
Diary. 15:26-7; Nash P. (2006) J Rheumatol. 33:1431-4. Responses to
the traditional disease-modifying anti-rheumatic drugs (DMARDs)
have been suboptimal. Kavanaugh et al. (2006) J Rheumatol.
33:1417-21; Soriano et al. (2006) J Rheumatol. 33:1422-30.
Anti-tumor necrosis factor (TNF) therapies are efficacious for both
skin and joint diseases but approximately 40% of subjects treated
with anti-TNF agents do not reach a minimal improvement in joint
responses (ACR20) and a large portion of subjects do not reach
higher level responses (i.e. ACR50 or 70). Mease et al. (2000) The
Lancet. 356:385-90; Mease et al. (2004) Arthritis and Rheum.
50(7):2264-72; Genovese et al. (2007) J Rheumatol. 34:1040-50;
Mease et al. (2005) Arthritis and Rheum. 52:3279-89; Mease P.
(2007) Therapeutics and Clinical Risk Management. 3:133-48; Antoni
et al. (2005) Arthritis and Rheum. 52:1227-36; Antoni et al. (2008)
J Rheumatol. 35:869-876; Antoni et al. (2005) Ann Rheum Dis.
64:1150-7. Several effective RA therapies have not provided the
desired response in PsA and left the anti-TNF agents the major
class of approved biologic therapy for PsA. Mease et al. (2011)
Arthritis and Rheum. 63:939-948. Several agents under development
for PsA had good efficacy for psoriatic skin lesions but with less
optimal joint efficacy. Mease P. (2006) Bulletin of the NYU
Hospital for Joint Diseases. 64: 25-31; Weger W. (2010) British
Journal of Pharmacology. 160:810-20; Gottlieb et al. (2009) The
Lancet. 373:633-40.
[0674] Therefore, there is still a significant unmet need in PsA
for therapies that provide higher levels of efficacy in the joints
in a greater proportion of subjects especially with the additional
attributes of durability of effect over time, low immunogenicity, a
subcutaneous dosing regimen that may allow for less frequent
administration, and a risk benefit profile that remains
acceptable.
[0675] Introduction:
[0676] This example presents results from the 24-week double-blind
period for clinical study report (CSR) IM133004, a Phase 2b,
randomized, double-blind, placebo-controlled, dose-ranging,
multicenter study, followed by a long-term extension (LTE) in
subjects with psoriatic arthritis (PsA) as diagnosed by the
Classification Criteria for Psoriatic Arthritis (CASPAR) with
active disease, who had an inadequate response to nonsteroidal
anti-inflammatory drug (NSAIDs) and naive to or with inadequate
response to non-biologic disease-modifying anti-rheumatic drugs
(DMARDs).
[0677] Clazakizumab (BMS-945429) is a genetically engineered
humanized IgG1 anti-interleukin-6 monoclonal antibody (anti-IL-6
mAb) that is being developed by Bristol-Myers Squibb Co. (BMS) for
the treatment of rheumatoid arthritis (RA) and other non-oncology
related indications. Clazakizumab is also known as ALD-518 and is
being developed by Alder BioPharmaceuticals, Inc. (Alder) for use
in cancer patients.
[0678] Interleukin-6 is a soluble pleiotropic cytokine that plays a
critical role in the pathogenesis of many inflammatory conditions,
including RA.1 Interleukin-6 mediates various functions of multiple
cell types, both immune and non-immune. These functions include
cell proliferation and differentiation, cell activation, B-cell
secretion of antibodies, hepatocyte production of acute phase
proteins, and hematopoiesis. The clinical relevance of IL-6 in RA
is demonstrated by the approval of tocilizumab (Actemra.RTM.), a
monoclonal antibody that binds to the IL-6 receptor and blocks the
biologic activity of IL-6. Monthly intravenous (IV) administrations
of tocilizumab significantly improve signs and symptoms and
suppress joint damage in RA patients.
[0679] In contrast, clazakizumab binds to the soluble human IL-6
cytokine, rather than to the IL-6 receptor. Together with other
properties such as high potency and long half-life (.about.30
days), the characteristics of clazakizumab may result in a
differentiated clinical profile compared to tocilizumab or other
biologics.
[0680] In this study, clazakizumab was administered subcutaneously
(SC) in subjects with moderate to severe active rheumatoid
arthritis with inadequate response to methotrexate (MTX). This
dose-ranging, placebo/active-controlled study was designed to
compare the efficacy and safety of clazakizumab with MTX or
clazakizumab monotherapy to placebo on background MTX over 24
weeks. Given the significant unmet needs in PsA, this study in PSA
(IM133004) was designed to evaluate the safety and efficacy of SC
injection of clazakizumab in PsA.
[0681] Objectives--
[0682] The primary objective during the double-blind period was to
compare the efficacy of three doses of clazakizumab subcutaneous
(SC) versus placebo (PBO) as assessed by American College of
Rheumatology 20% improvement (ACR20) response rates at 16
weeks.
[0683] The secondary objectives of the double-blind period were to:
(i) assess additional efficacy outcomes of clazakizumab SC at 16
weeks as measured by psoriasis area and severity index (PASI;
specifically PASI75), ACR50 and ACR70 response rates, physical
function and health related quality of life outcomes; (ii) assess
efficacy outcomes of clazakizumab SC at 24 weeks as measured by
PASI75, ACR20, ACR50 and ACR70 response rates, physical function
and health related quality of life outcomes; and (iii) assess
safety, tolerability and immunogenicity of clazakizumab SC
injections.
[0684] Methodology--
[0685] This clinical study report presents results from the 24-week
double-blind period for Study IM133004 (incorporated herein by
reference), a Phase 2b, randomized, double-blind,
placebo-controlled, dose-ranging, multicenter study, followed by an
long-term extension (LTE) in subjects with psoriatic arthritis
(PsA) by the Classification Criteria for Psoriatic Arthritis
(CASPAR) with active disease, who had an inadequate response to
nonsteroidal anti-inflammatory drug (NSAIDs) and/or non-biologic
disease-modifying anti-rheumatic drugs (DMARDs).
[0686] Period I (Randomization/Day 1 to Week 16):
[0687] Upon meeting the inclusion/exclusion criteria, subjects were
randomized to 1 of the 4 treatment arms (placebo or clazakizumab 25
mg SC every 4 weeks, 100 mg SC every 4 weeks, or 200 mg SC every 4
weeks) with a 1:1:1:1 ratio as shown in FIG. 16.
[0688] After the study began, that FDA mandated that all subjects
be on active treatment to avoid the potential for radiographic
progression over 24 weeks in subjects randomized to placebo. The
protocol was amended to attempt to put all subjects on methotrexate
(MTX) and to standardize the MTX treatment by placing such subjects
on MTX at Week 16. Subjects who were not on MTX between Weeks 0 and
16 were allowed to continue without MTX until Week 16 when they
were all placed on MTX.
[0689] Period II (Week 16 to Week 24):
[0690] All subjects who completed Period I continued to receive the
same treatment assignments during Weeks 16 to 24. Subjects who had
been enrolled in this study but were not yet randomized, and who
were not on a background of MTX, were placed on oral MTX 15 mg/week
(but no less than 10 mg/wk) at Week 16; this allowed subjects
randomized to placebo to be placed on active therapy. Those
subjects (who enrolled early in the study) who were between Weeks 0
and 16 and who were not on MTX were allowed to continue without
receiving MTX until Week 16 when they were all placed on MTX.
Subjects who were after Week 16 by the time of the FDA mandate (for
active treatment) and were not already on MTX were allowed to go on
without MTX to Week 24 when they started receiving clazakizumab 200
mg as per protocol.
[0691] During Period II, doses of oral glucocorticosteroids could
be changed but the total dose remained .ltoreq.10 mg/day of
prednisone or prednisone equivalent. NSAID dose changes were also
allowed according to investigator's clinical judgment for
appropriate disease management.
[0692] In addition, subjects that did not achieve at least a 20%
reduction compared to baseline in the swollen and tender joint
count during Weeks 16 to 24, and in the LTE until the switch to the
final dose of 25 mg clazakizumab, were eligible for rescue
therapy.
[0693] Number of Subjects (Planned and Analyzed):
[0694] 168 subjects were planned to be included (42 subjects per
arm); 165 subjects were analyzed (41 each in the placebo, 25 mg
clazakizumab, and 200 mg clazakizumab groups and 42 subjects in the
100 mg clazakizumab group).
[0695] Diagnosis and Main Criteria for Inclusion:
[0696] Subjects must have had a diagnosis of PsA by CASPAR criteria
and had active disease for at least 12 weeks prior to screening.
Subjects must have had inadequate responses to NSAIDs and/or
non-biologic DMARD therapy. Subjects must have had a minimum of
>3 swollen and >3 tender joints (66/68 joint counts); active
psoriatic skin lesions of >3% Body Surface Area (BSA); and a
high sensitivity CRP (hsCRP) of >ULN (by central laboratory
values) at screening. Subjects who were on MTX were allowed if they
had been taking MTX for at least 3 months at a dose >15 mg/week
to a maximum weekly dose of .about.25 mg, and were at a stable dose
of MTX for 4 weeks prior to randomization (Day 1). Other
non-biologic DMARDs had to be washed out according to the
protocol.
[0697] Subjects are excluded if they had previously received or
were currently receiving an approved biologic therapy for PsA or
psoriasis. Subjects were excluded if they had active systemic
inflammatory condition other than PsA which might have interfered
with the results of clinical or laboratory tests planned in the
study (eg, systemic lupus erythematosus or any other systemic
rheumatic disease other than PsA).
[0698] Criteria for Evaluation: Efficacy:
[0699] Clinical joint, skin, dactylitis, and enthesitis assessments
were conducted. The primary efficacy assessment was the proportion
of subjects meeting the ACR criteria for improvement (ACR20) at
Week 16. The secondary efficacy assessments comprised: individual
components of the ACR core data set, ACR50, ACR70, PASI75, Health
Assessment Questionnaire--Disability Index (HAQ-DI), Short Form
(36) (SF-36). Safety: The evaluation of drug safety was based on
clinical AEs, vital signs, ECGs and laboratory abnormalities
reported during the double-blind study period. Immunogenicity:
Serum samples were assayed for the presence of anti-clazakizumab
antibodies.
[0700] Statistical Considerations:
[0701] For the primary endpoint of ACR20 at Week 16, assuming the
placebo response rate of 15% with an a=0.017 (two-sided),
approximately 42 subjects per arm (total 168) would provide around
85% power to detect a difference in treatment response rate of
37%.
[0702] The primary testing procedure involved 3 comparisons of
clazakizumab (25 mg SC every 4 weeks, 100 mg SC every 4 weeks, or
200 mg SC every 4 weeks) versus placebo and Dunnett-Tamhane step-up
procedure was performed to control the overall type I error rate at
0.05 for multiple comparisons.
[0703] Efficacy Results:
[0704] Overall Efficacy Summary:
[0705] The study met its primary objective of at least one dose of
clazakizumab being statistically superior to placebo on the primary
endpoint of ACR20 at Week 16.
[0706] The proportion of subjects with an ACR20 response rate at
Day 113 (Week 16) was numerically higher in the 3 clazakizumab
groups compared with the placebo group (Table 14 and FIG. 17). In
the 100 mg clazakizumab group, this improvement (difference of
23.1%) was statistically superior to placebo (adjusted
p-value=0.039; Table 14).
TABLE-US-00014 TABLE 14 Proportion of Subjects Achieving ACR20 at
Day 113 (Week 16) (All Randomized and Treated Subjects) PBO +/- MTX
B25 +/- MTX B100 +/- MTX B200 +/- MTX N = 41 N = 41 N = 42 N = 41 #
OF SUBJECTS <Y/X> (%) 12/41 (29.3) 19/41 (46.3) 22/42 (52.4)
16/41 (39.0) 95% CI (15.3, 43.2) (31.1, 61.6) (37.3, 67.5) (24.1,
54.0) ESTIMATE OF DIFFERENCE (%) 17.1 23.1 9.8 95% CI (-3.6, 37.7)
(2.6, 43.7) (-10.7, 30.2) P-VALUE (VS PLACEBO) 0.101 0.039 0.178 Y
= Number of subjects with measure/event of interest, X = Number of
subjects in the analysis. For CI within each group, normal
approximation is used if Y >= 5 and X - Y >= 5. Otherwise
exact method is used. For CI of difference, normal approximation is
used if Y >= 5 and X - Y >= 5 in both arms. Otherwise exact
method is used. P-value is based on Dunnett-Tamhane step up
procedure. ACR20 = 20% ACR response; PBO = Placebo; MTX =
Methotrexate; CI = Confidence interval.
[0707] No dose response for efficacy was noted in this study; the
25 mg and 100 mg clazakizumab doses tended to show better efficacy
results for most parameters compared to the 200 mg clazakizumab
dose group and placebo.
[0708] When examining efficacy result for the 5 efficacy domains of
PsA, the benefits of clazakizumab treatment were shown as
follows:
[0709] Joints--improvement was noted in tender and swollen joints
as shown by positive results of the ACR20, ACR50, ACR70 (FIGS.
18-20, respectively), individual components of the American College
of Rheumatology (ACR) (FIG. 21 for tender joint count and FIG. 22
for swollen joint count), DAS28-CRP (Disease Associated Score,
C-reactive protein, data not shown), DAS28-CRP <2.6 (data not
shown), HAQ-DI (Health Assessment Questionnaire, Disability Index,
data not shown), and the PsARC (composite index that is
predominantly associated with outcomes related to changes in tender
and swollen joints, data not shown). Clazakizumab showed early
(onset of action by 4 weeks) and consistent improvement over
placebo in these joint measures with greater improvement more often
being seen in the 25 mg and 100 mg clazakizumab doses compared to
the 200 mg dose at Day 113 (Week 16) and Day 169 (Week 24).
[0710] Skin--there was no difference in the PASI75 (Psoriasis Area
Severity Index) between any of the clazakizumab doses and placebo
at Day 113 (Week 16); however, there appeared to be a marginal
improvement over placebo in both the 25 and 100 mg clazakizumab
dose groups on the PASI75 and PASI50 response at Day 169 (Week 24).
No consistent results were noted when comparing mean change from
baseline in PASI over time (data not shown).
[0711] Enthesitis--there was apparent improvement in enthesitis
with clazakizumab treatment compared with placebo. The SPARCC
Enthesitis Index showed that the proportion of subjects with
enthesitis at baseline decreased over time in the 3 clazakizumab
dose groups compared with placebo at both Day 113 (Week 16) and Day
169 (Week 24). There was no meaningful difference in the mean
change from baseline LEI (Leeds Enthesitis Index) score among the 3
clazakizumab groups and the placebo group (data not shown).
[0712] Dactylitis--the results for this parameter based on the LDI
(Leeds Dactylitis Index) were not able to be analyzed for this
study due to unreliable data collection for this measure. However,
in the clazakizumab groups there was an apparent decrease over time
in tender and swollen digits among those subjects with at least 1
tender and swollen digit (data not shown).
[0713] Spine--In this study, the BASDAI (Bath Ankylosing
Spondylitis Index) score was used as a surrogate measure of spinal
involvement; in the 25 and 100 mg clazakizumab groups showed a
small numerical improvement over placebo on this scale (data not
shown).
[0714] Safety Results:
[0715] Overall Safety Summary:
[0716] Clazakizumab was tolerated at all doses studied. Few
subjects discontinued therapy over the first 24 weeks of the study.
Nevertheless, the study data did reveal dose-related safety
findings. Overall, the 25 mg clazakizumab dose group had the lowest
frequency of AEs, discontinuation due to AEs, transaminase
abnormalities and injection site reactions whereas a higher
incidence of these safety measures was noted in the 200 mg group.
Although the safety profile of the 100 mg clazakizumab dose group
was similar in many respects to the 25 mg dose group, the
benefit-risk profile of clazakizumab appeared most favorable at 25
mg. No new safety signals were observed compared to the RA patients
treated with clazakizumab. No subject died during the study and no
subject had laboratory abnormalities that met the criteria for Hy's
Law.
[0717] Safety data were available for 165 subjects in the
As-Treated Analysis Population including those subjects who
received at least 1 dose of double-blind treatment. This dataset
included the 149 subjects who completed through Week 16 of the
study (Period I) and the 140 subjects who completed Weeks 16 to 24
(Period II). Safety observations for the double-blind treatment
period include results from Period I (through Week 16; data not
shown) and results from both Periods I and II (through Week 24;
data not shown). Safety observations for Periods I and II
(cumulative through Week 24) are summarized as follows:
[0718] Deaths:
[0719] No subject died during the double-blind treatment period of
the study.
[0720] Serious Adverse Events (SAEs):
[0721] Serious adverse events were reported for 10 (6.1%) subjects
(including 7 [4.2%] subjects in Period I): 2 subjects (4.9%) in the
placebo group, 2 subjects (4.9%) in the 25 mg clazakizumab group, 2
subjects (4.8%) in the 100 mg clazakizumab group, and 4 subjects
(9.8%) in the 200 mg clazakizumab group. With the exception of
transient hypoesthesia and muscular weakness in Subject
IM133004-70-103 in the 200 mg clazakizumab group, none of these
SAEs was reported by the investigator to be related to study drug.
In the placebo group, SAE PTs were bladder neoplasm and
transitional cell carcinoma in 1 subject and prostate cancer in 1
subject. In the 25 mg clazakizumab group, SAE was bradycardia and
chest pain each in 1 subject. In the 100 mg clazakizumab group, SAE
was psoriasis and dystonia each in 1 subject. In the 200 mg
clazakizumab group, SAEs were hypoesthesia and muscular weakness in
1 subject, and intervertebral disc disorder, acute myocardial
infarction, and hemiparesis each in 1 subject.
[0722] Discontinuations Due to Adverse Events (AEs):
[0723] Thirteen (7.9%) subjects discontinued due to AEs including 2
subjects in the placebo group who discontinued due to SAEs of
transitional cell carcinoma and prostate cancer, and 1 subject in
the 200 mg clazakizumab group who discontinued due to SAEs of
hypoesthesia and muscular weakness. Eight (4.8%) of these subjects
discontinued during Period I.
[0724] Adverse Events of Special Interest:
[0725] Infections and infestations (including nasopharyngitis,
pharyngitis, urinary tract infection [UTI], upper respiratory tract
infection [URTI], and bronchitis) were noted in a higher proportion
of subjects in the placebo group (48.8%) compared to the 3
clazakizumab groups (36.6%, 35.7%, and 24.4%, in the 25 mg, 100 mg
and 200 mg groups, respectively). There were no cases of
tuberculosis or reports of opportunistic infections. Hepatic
disorders (including increased ALT and AST) were only reported in
the 3 clazakizumab groups (22.0%, 16.7%, and 26.8%, in the 25 mg,
100 mg and 200 mg groups, respectively). There was a low incidence
(<10% of subjects) of local injection site events in the 25 mg
(9.8%) and 100 mg (9.5%) clazakizumab groups and in the placebo
group (4.9%); local injection site events were reported with higher
frequency in the 200 mg clazakizumab group (17.1%). No subject
experienced autoimmune disorders, systemic injection events,
demyelinating disorders or gastrointestinal perforations during the
double-blind treatment period. There were two malignancies reported
in the placebo group (transitional cell carcinoma and prostate
carcinoma) but no malignancies were reported in the 3 clazakizumab
groups.
[0726] Overall AEs:
[0727] AEs were reported for 123 (74.5%) subjects (including 115
[60.7%] subjects during Period I) and AEs were assessed as
drug-related for 71 (43.0%) subjects (including 64 [38.8%] subjects
during Period I). The highest incidence of AEs (82.9%) and related
AEs (63.4%) were noted in the 200 mg clazakizumab group. Adverse
events reported for >5% of subjects overall included increased
ALT (13.9%), increased AST (8.5%), nasopharyngitis (7.9%),
hypercholesterolemia (7.3%), pharyngitis (6.1%), URTI (6.1%),
headache (6.1%), and hypertension (6.1%).
[0728] Laboratory Abnormalities:
[0729] With a few exceptions, changes from baseline in most
laboratory values were similar across the treatment groups. Changes
from baseline that were more prevalent in the clazakizumab groups
compared with the placebo group included decreases in mean absolute
neutrophil count, decrease in mean platelet count, increases in
total cholesterol levels, increases in mean ALT (Alanine
aminotransferase), AST (Alkaline phosphatase), and total bilirubin
levels, and decreases in mean alkaline phosphatase levels.
[0730] Vital Signs and ECG:
[0731] No safety concerns were identified based on evaluation of
laboratory and vital sign data.
[0732] Pharmacokinetic Results:
[0733] The following is a brief summary of the key pharmacokinetic
findings: The degree of fluctuation between the maximal and minimal
(pre-dose) concentration was minimal (less than 2 fold) across dose
range following SC administration of clazakizumab. Based on
observed trough (Cmin) concentrations, steady-state was reached by
the time of Week 20 which is consistent with the half-life of
clazakizumab. Following the SC dose of clazakizumab in the range of
25 mg to 200 mg, Cmax and AUC(TAU) (Area under the serum
concentration-time curve over a dosing interval, TAU=4 weeks)
increased slightly less than proportionally to dose, which may be
attributed to limitations in sample size and variability in sample
collection times. Median Tmax values were 3 to 7 days (ranged 2 to
21 days) across the treatment groups (data not shown).
[0734] Pharmacodynamic Results:
[0735] As an indicator of target engagement, total (FIG. 23) and
free (FIG. 24) levels of serum IL-6 were measured using validated
immunoassays that discriminate between clazakizumab bound IL-6
(total IL-6) vs. non-clazakizumab bound ligand (free IL-6). At
baseline, levels of IL-6 ranged between 10 to 25 pg/mL (Table
S.9.3). In the placebo arm, there was little change in total or
free IL-6 throughout the course of the study. In the clazakizumab
treated subjects, free IL-6 decreased below the level of detection
(3.6 pg/mL) immediately postdosing and across all dose groups.
These changes were observed at the earliest time point (Day 8) and
were sustained throughout the study (Day 169 [Week 24]). For the
200 mg clazakizumab dose group, mean levels of free IL-6
demonstrated detectable levels of free IL-6 at Days 8 and 29,
however, this result was due to 1 outlier that had delayed
suppression of IL-6 levels. Upon further treatment, this subject
showed undetectable levels of free IL-6 starting at Day 57 that
were maintained through Day 169. Postdosing with clazakizumab,
there was a rapid increase in total IL-6 levels reflective of the
increase in drug bound ligand. Total IL-6 levels increased
approximately 200-fold above baseline at Day 8 (mean range 2355 to
3832 pg/mL) and appeared to plateau at later time points to
approximately 500-fold above baseline (mean range 7175 to 12209
pg/mL at Days 113 and 169). An observed increase in the total IL-6
levels in the 200 mg dose group at Day 113 was driven by a few
outliers and was not representative of the entire group. There was
a modest dose response that was observed across the dose groups
with a lower increase in total IL-6 levels in the 25 mg
clazakizumab dose group compared to the two higher clazakizumab
dose groups (100 and 200 mg). Overall, these results demonstrate
target engagement of clazakizumab treatment at all dose groups and
throughout the course of the study.
Example 22
Phase III, Randomized, Multi-Center, Double-Dummy, Double-Blind,
Placebo and Active-Comparator Study to Evaluate the Efficacy and
Safety of Clazakizumab Compared to Placebo and Adalimumab in
Subjects with Active Rheumatoid Arthritis Who are Inadequate
Responders to Oral Conventional Synthetic Disease Modifying
Anti-Rheumatic Drugs (DMARDs)
[0736] Herein we provide clinical regimens using low dosing
regimens which further validate the improved clinical benefit of
the subject anti-IL-6 antibody comprising the heavy and light
polypeptides of SEQ ID NO:709 and 657 (clazakizumab) relative to
established RA therapies in achieving a high level of disease
control using a stringent measure of signs and symptoms (i.e. DAS
28 CRP <2.6), along with clinically meaningful benefits in
physical function and inhibition of structural damage, and a
favorable overall safety profile in patients who are inadequate
responders to conventional synthetic DMARDs or naive to MTX.
[0737] Particularly, these studies will confirm the efficacy and
safety of SC clazakizumab compared to placebo and to adalimumab in
combination with MTX, in subjects with moderate to severe RA who
have IR to at least one conventional synthetic DMARD including MTX.
Data from this study will demonstrate that clazakizumab is superior
to adalimumab in the proportion of patients who achieve a high
level of disease control, as measured by DAS 28 CRP <2.6.
Efficacy
[0738] The subject anti-IL-6 antibody comprising the variable heavy
and light polypeptides of SEQ ID NO:709 and 657 (clazakizumab) when
used at doses of 25 mg, 100 mg, and 200 mg/month with background
MTX, 100 mg and 200 mg monotherapy) demonstrated efficacy over
placebo. In addition, each of the clazakizumab+MTX doses was
associated with more patients achieving stringent measures of
response than with adalimumab+MTX. Overall, there was not a strong
dose response relationship at the doses tested on ACR20, ACR50,
ACR70, DAS28-CRP<2.6, CDAI<2.8, or SDAI<3.3. These
findings were corroborated by exposure response analyses, which
examined the relationship between steady-state C.sub.min
concentrations (Cmins), and ACR response rates. Within the range of
Cmins achieved with the 25 mg and higher, the relationship between
Cmins and the probability of achieving an ACR response was
relatively flat (FIG. 25). While the overall magnitude of response
was lower for monotherapy regimens compared to the MTX combination
therapies, the relationship between C.sub.mins and the probability
of achieving ACR response was again flat across the exposures
associated with the 100 and 200 mg monotherapy doses. Exposure
response analyses for alternate efficacy endpoints, including DAS
28 CRP reduction and CDAI reduction (which excludes CRP) were
similar.
[0739] In these clinical trials RA patients are treated with low
doses (1 mg, 5 mg, and 25 mg/month of clazakizumab with background
MTX) to confirm the optimal benefit/risk of the 25 mg clazakizumab
dose in RA patients with similar disease activity and is expected
to have a more favorable benefit-risk profile than the lower doses.
Therefore, clazakizumab 25 mg SC q 4 weeks is planned to be the
dose used in this study.
Safety
[0740] Clazakizumab was well-tolerated and efficacious (resulted in
remission of disease symptoms) at all doses studied. Nevertheless,
Phase 2b data did reveal some dose-related safety findings.
However, it was observed that the clazakizumab 25 mg+MTX group had
the lowest frequency of AEs, discontinuation due to AEs, LFT
abnormalities and injection site reactions among the 3 MTX
combination treatment groups studied and yet was still effective in
treating disease symptoms. There were no substantive differences in
the safety profiles of the 100 and 200 mg monotherapy treatment
groups. In addition, with the exception of LFT abnormalities, there
was no meaningful difference in the safety profile of clazakizumab
administered as combination vs as monotherapy. These results
suggest that the safety profile of clazakizumab is acceptable at
all doses studied, but is most safe and effective at 25 mg or lower
monthly dosages.
a) Study Rationale
[0741] Based thereon the current study aims to demonstrate clinical
benefits of clazakizumab, when used in combination with MTX, in RA
patients with moderately to severely active disease, who are
inadequately responding to conventional synthetic DMARDs. Many
elements of the study are based on prior study designs for studying
new drugs in RA for registrational purposes. The chosen patient
population is appropriate to study the efficacy and safety of
clazakizumab in keeping with the treat-to-target strategy that
early biologic intervention use in appropriate patients may prevent
the long-term structural damage observed with inadequate early
control. The confirmatory Phase 3 study is also intended to
demonstrate that clazakizumab has clinically beneficial effects on
other measures of signs and symptoms and physical function in
patients with moderate to severe RA. The study contains placebo and
active (adalimumab) comparators. Inclusion of the placebo arm will
allow for ensuring assay sensitivity of effect size and adalimumab
is chosen as an active comparator as it is the biologic standard of
care agent in this population. Placebo duration of 12 weeks allows
for determination of the effect size of clazakizumab at an
appropriate time interval while permitting adequate management of
symptoms in patients that are not meeting therapeutic goals.
Comparison of clazakizumab to adalimumab will occur at 24 weeks, a
time point by which it is expected that both clazakizumab and
adalimumab will have achieved maximal efficacy to allow for
assessment of the higher efficacy measures (i.e. DAS CRP
<2.6).
[0742] The long term extension of the study is intended to evaluate
the long term safety of treatment with clazakizumab as well as the
durability of the clinical response to clazakizumab. During the
long term extension, subjects on adalimumab will be switched to
clazakizumab to determine if there is a further improvement in
signs and symptoms of RA in those subjects.
b) Dose Selection Rationale
[0743] RA subjects who are inadequate responders to synthetic
DMARDs (including MTX) and have moderate to severely active
disease, who were clazakizumab 25 mg demonstrated the best
benefit-risk profile compared to other tested doses. Based thereon
clazakizumab is administered 25 mg SC q 4 weeks.
Pharmacodynamic Data
[0744] As an indicator of target engagement, inhibition of the
soluble IL-6 and soluble IL-6 receptor complex formation was
derived using a validated method that incorporates the direct
measurements of clazakizumab concentration, total IL-6
concentrations, and binding affinity of clazakizumab to IL-6. When
assessing IL-6/IL-6 soluble receptor complex inhibition across
clazakizumab doses, there were differences across dose arms, such
that higher doses exhibited higher distributions of IL-6/IL-6
soluble receptor complex inhibition. Examination of the
clazakizumab IL-6/IL-6 soluble receptor complex inhibition data by
ACR20 response, suggests that ACR20 responders had higher levels of
inhibition at Week 12 than non-responders with the 25 mg dose in
combination with MTX (FIG. 26). Similar trends were observed with
ACR50 and ACR70. A validated enzyme linked immunosorbent assay
(ELISA) method is used to measure concentrations of clazakizumab in
serum.
[0745] Trough concentrations (Cmin) of clazakizumab will be
summarized by visit day. Collected PK data will be combined with
data from other studies for population PK analysis. This analysis
will examine the potential effects of covariates such as age, body
weight, ethnicity etc. on PK. Exposure-Response relationship
between measures of exposure and selected efficacy (e.g., ACR,
DAS28-CRP) and safety (eg, liver abnormality tests) endpoints will
be characterized. Results from these analyses will be reported
separately.
Immunogenicity and Biomarkers
[0746] Predose serum samples will be collected at baseline (predose
Day 1), at specified time points during the double-blind period and
long term extension period, as well as at specified times after the
subjects discontinues from the study.
[0747] Samples are assayed for the presence of anti-clazakizumab
antibodies using a validated ECL assay method. The incidence of the
formation of anti-clazakizumab antibodies will be summarized by
treatment. The effect of anti-clazakizumab antibodies on the
systemic exposure, safety, and efficacy of clazakizumab will be
evaluated.
Objectives:
[0748] These clinical studies will compare the efficacy of
Clazakizumab versus PBO, both on background MTX, in terms of
reducing signs and symptoms of RA as assessed by proportion of
subjects achieving ACR20 at 12 weeks of treatment.
[0749] Also, these clinical studies will evaluate the following:
[0750] 1) the efficacy of Clazakizumab versus PBO, both on
background MTX, in improving physical function in RA as assessed by
change in HAQ-DI over baseline at 12 weeks of treatment. [0751] 2)
the efficacy of Clazakizumab versus PBO, both on background MTX, in
achieving low disease activity as assessed by proportion of
subjects with DAS28-CRP<2.6 at 12 weeks of treatment; [0752] 3)
the efficacy of Clazakizumab versus Ada, both on background MTX, in
achieving low disease activity as assessed by proportion of
subjects with DAS28-CRP<2.6 at 24 weeks of treatment; and [0753]
4) the efficacy of Clazakizumab versus Ada, both on background MTX,
in reducing RA signs and symptoms as assessed by proportion of
subjects with ACR70 at 24 weeks of treatment.
[0754] Further, these clinical studies will evaluate the following:
[0755] 1) efficacy responses (ACR20/50/70; HAQ-DI; SDAI.ltoreq.3.3;
CDAI.ltoreq.2.8; DAS28-CRP<2.6; DAS28-ESR<2.6; change from
baseline of DAS28-CRP; change from baseline of DAS28-ESR) over 24
weeks; [0756] 2) safety of Clazakizumab on background MTX through
assessments of adverse events (AEs) and laboratory parameters; and
[0757] 3) systemic exposure, immunogenicity and pharmacodynamics
(PD) of Clazakizumab on background MTX.
[0758] Still further, these clinical studies will evaluate the
following: [0759] 1) the long-term maintenance of efficacy
responses (ACR20/50/70; HAQ-DI; SDAI.ltoreq.3.3; CDAI.ltoreq.2.8;
DAS28-CRP<2.6; DAS28-ESR<2.6; change from baseline of
DAS28-CRP; change from baseline of DAS28-ESR) of Clazakizumab on
background MTX beyond 24 weeks; [0760] 2) the median time to onset
of efficacy measures of Clazakizumab on background MTX; [0761] 3)
the efficacy of Clazakizumab on background MTX in OLE in subjects
who had received Ada in the double-blind period; [0762] 4) the
efficacy of Ada versus PBO both on background MTX in achieving
DAS28-CRP<2.6 over 12 weeks of treatment. [0763] 5) the efficacy
of Clazakizumab on background MTX on Quality of Life measure
(SF-36). 6) work productivity (WPAI-RA) with Clazakizumab versus
PBO over 12 weeks and versus Ada over 24 weeks, all on background
MTX; [0764] 7) fatigue (FACIT) with Clazakizumab versus PBO over 12
weeks and versus Ada over 24 weeks, all on background MTX; [0765]
8) the effects of covariates on the PK of Clazakizumab on
background MTX and evaluate the exposure-response relationship for
efficacy, safety, and PD markers (e.g. CRP, total IL-6, and free
IL-6); and [0766] 9) biomarkers (including soluble, intracellular,
and genomic) which may be used to predict and monitor treatment
response and safety associated with treatment with Clazakizumab or
Ada.
Study Design and Duration:
[0767] Subjects with moderate to severe rheumatoid arthritis, who
are inadequate clinical responders to conventional synthetic DMARDs
(upon meeting the requirements based on inclusion and exclusion
criteria), are randomized into 1 of the 3 treatment arms as shown
in FIG. 26.
Screening
[0768] Upon obtaining the informed consent, a subject's eligibility
will be determined. Subjects must have experienced an inadequate
clinical response to one or more conventional synthetic DMARDs
(which must include MTX) as documented by a treating physician or
investigator (see Section 3.3.1 for definition of inadequate
response).
[0769] All subjects must have been receiving treatment with a
minimum dose of 15 mg per week (maximum 25 mg per week) of
methotrexate for at least 12 weeks and at a stable dose for 6 weeks
prior to randomization. A lower dose of methotrexate is permitted
in some circumstances Also, to minimize potential methotrexate
toxicity all subjects should receive folic acid, folinic acid, or
leucovorin according to the manufacturer recommendations and the
local medical standard of care guidelines.
[0770] Oral prednisone or equivalent is permitted, if the dose is
10 mg/day and if it has been stable for 4 weeks before screening.
Non-steroidal anti-inflammatory drugs (NSAIDs) must be stable for 4
weeks before screening and consistent with labeling
recommendations. IA, IV and IM corticosteroid injections may not be
administered within 4 weeks of screening.
Period 1: Double-Blind/Placebo Controlled Period; Randomization to
Week 12 (Primary Endpoint)
[0771] Following the screening period, eligible subjects will be
randomized to 1 of 3 parallel arms on Day 1 at a 2:2:1 ratio as
follows:
Period 1 Treatment Arms:
[0772] 1. Clazakizumab 25 mg SC every 4 weeks [0773] PLUS Placebo
for adalimumab SC every 2 weeks with background methotrexate
(n=460) 2. Adalimumab 40 mg SC every 2 weeks [0774] PLUS Placebo
for clazakizumab SC every 4 weeks with background methotrexate
(n=460)
3. No Active Treatment
[0774] [0775] Placebo for adalimumab SC every 2 weeks [0776] PLUS
Placebo for clazakizumb SC every 4 weeks with background
methotrexate (n=230)
[0777] Subjects and caregivers are trained during the first two
study visits of this period in self administration of the study
medication using pre-filled syringes. All subsequent injections
will be self administered or administered by a caregiver, not by
the physician or medical staff at the study site. During this
period, the dose of methotrexate, NSAIDs, and oral prednisone (or
its equivalent) should remain stable. Intra-articular
corticosteroid injections and intramuscular corticosteroid
injections are not permitted. Analgesics are permitted with certain
restrictions (See Restricted and Prohibited medications)
Period 2: Double-Blind/Active Drug Period; Week 12 to Week 24
[0778] During this period, subjects assigned to Treatment Arm #3
(no active treatment) will switch to the active study drug regimen
described in Treatment Arm #1 (clazakizumab 25 mg SC every 4
weeks). For these subjects, concomitant medication requirements
will not change. In all treatment arms, subjects will receive their
final dose of study drug for this period at week 24.
Period 3: Long Term Extension (LTE)
[0779] Subjects who continue to demonstrate clinical benefit at the
end of Period 2, may elect to enter the LTE period. Subjects
assigned to the Treatment Arm #2 will receive clazakizumab placebo
at the LTE Day 1 and LTE Wk 4 visits. Following this washout
period, subjects assigned to this arm will receive active
clazakizumab 25 mg every 4 weeks. Subjects assigned to Treatment
Arms #1 and #3 will receive active clazakizumab 25 mg every 4 weeks
beginning with the LTE Day 1 visit.
[0780] Methotrexate (up to 25 mg), oral prednisone (.ltoreq.10
mg/day) or its equivalent, NSAIDs and analgesics may be adjusted at
the investigator's discretion during the LTE period. A single
course of high dose oral, IM, or IA corticosteroid injection is
permitted every six months.
[0781] The LTE will continue as an open-label study up to 12 months
after the approval of study drug by the responsible health
authority or until it becomes commercially available within the
country, whichever occurs sooner. During the long term extension
all subjects who continue to demonstrate clinical benefit as
determined by their study physician will be eligible to continue to
receive study drug. It is possible that the study drug dosing
regimen will change during the long term extension or that subjects
may be offered enrollment in another study or drug access program.
If this occurs, details will be provided in the form of an
amendment to the protocol.
Post-Study Drug Follow-Up Period (6 Months)
[0782] Subjects who discontinue treatment of study drug or complete
the study will have follow-up visits for up to six months, to
perform safety and laboratory assessments. During this period, when
subjects are no longer receiving study drug, it is recommended that
they not be treated with other biologic therapy, due to
clazakizumab's half-life of around 30 days. However, this decision
remains at the investigator's discretion. If the study drug becomes
commercially available, and if the subject chooses to receive
treatment with the commercial product, then this period is not
required.
Key Inclusion/Exclusion Criteria:
[0783] Adult patients with RA for at least 16 weeks who have had a
clinically inadequate response to conventional synthetic DMARDs
including MTX but who have not used biologic therapy. Subjects must
have clinical and laboratory evidence of active, moderate to severe
RA. Subjects are excluded for high risk of infection, liver
dysfunction, GI inflammation, and the presence of other non-RA
rheumatologic disease.
Inclusion Criterion:
[0784] a) documented diagnosis of active RA by standard criteria
(ACR/EULAR [2010]) at least 16 weeks prior to screening; [0785] b)
ACR global functional status class of 1 to 3; [0786] c) Documented
evidence of inadequate clinical response to one or more
conventional synthetic DMARDs (which must include MTX); [0787] d)
Methotrexate and conventional synthetic DMARDs: All subjects must
have been receiving treatment with a minimum dose of 15 mg per week
(maximum dose of 25 mg per week) of methotrexate for at least 16
weeks and at a stable dose for 6 weeks prior to randomization. A
lower dose of methotrexate dose is permitted if there is verifiable
documentation in the medical record prior to entry into the study
that the subject could not receive or reach a dose of 15 mg due to
toxicity and the dose is at least 10 mg methotrexate at the time of
screening. In countries where the standard of care requires use of
a lower doses of methotrexate (e.g. Japan), a minimum dose of 7.5
mg per week is permitted (to minimize potential methotrexate
toxicity, all subjects must receive folic acid, folinic acid, or
leucovorin according to manufacturer recommendations and local
medical standard of care guidelines; [0788] e) minimum of 6 swollen
and 6 tender joints on a 66/68 joint count at screening and at
baseline (Day 1); [0789] f) subjects must have at least one of the
following values at the screening: [0790] i. hsCRP of 0.8 mg/dL (8
mg/L) as measured by the Central Laboratory [0791] ii. Erythrocyte
Sedimentation Rate (ESR) 28 mm/h ExclusionCriterion: 1. Target
Disease Exceptions a) Subjects with documented juvenile rheumatoid
arthritis.
2. Medical History and Concurrent Diseases
[0791] [0792] a) Subjects at risk for tuberculosis (TB).
Specifically, subjects with: [0793] i. Current clinical,
radiographic or laboratory evidence of active TB. Chest x-rays (PA
and lateral) obtained within the 3 months prior to randomization
will be permitted but the images must be available and reviewed by
the investigator. TB testing (IFNg release assay or PPD) performed
in the past month prior to randomization will be accepted, however
a copy of the report must be placed in the subject binder. [0794]
ii. A history of active TB unless there is documentation that the
prior anti-TB treatment was appropriate in duration and type.
[0795] iii. Treatment for latent TB per local guidelines or at
minimum one of the following antimicrobial regimens (whichever is
longer) which has not yet been completed. [0796] INH 300 mg daily
for 6 months [0797] Rifampicin 600 mg daily for 4 months [0798] INH
300 mg daily+Rifampicin 600 mg daily for 3 months [0799]
Directly-observed rifapentine 900 mg plus INH 900 mg weekly for 3
months [0800] b) Subjects with any acute infection within the last
60 days prior to screening that required hospitalization or
treatment with parenteral antibiotics. Subjects with any acute
infection within the last 30 days prior to randomization that
required oral antimicrobial therapy. Subjects with active infection
at the time of randomization will be excluded. [0801] c) Subjects
with history of chronic or recurrent bacterial infection (such as
chronic pyelonephritis, osteomyelitis and bronchiectasis etc.)
[0802] d) Subjects who have a history of systemic fungal infections
(such as histoplasmosis, blastomycosis, or coccidiomycosis etc.).
[0803] e) Subjects with history of recurrent herpes zoster (more
than one episode) or recurrent herpes simplex (more than 2 episodes
per year) outbreaks or disseminated (more than one dermatome)
herpes zoster or disseminated herpes simplex will be excluded.
Symptoms of herpes zoster or herpes simplex must have resolved more
than 60 days prior to randomization. [0804] f) Subjects with
history of Human Immunodeficiency Virus (HIV) infection or who test
positive for HIV at screening. [0805] g) Subjects with history of
primary or secondary immunodeficiency or a family history of a
primary immune deficiency in a first degree relative. [0806] h)
Subjects with autoimmune disease other than RA (eg, SLE, multiple
sclerosis [MS], vasculitis, seronegative spondyloarthritis,
Inflammatory Bowel Disease etc.). However, patients with secondary
Sjogren's syndrome will be allowed. Subjects with active
fibomyalgia will be excluded. [0807] i) Prior history of or current
inflammatory joint disease other than RA (eg, gout, reactive
arthritis, Lyme's disease etc.). [0808] j) Subjects who are not
appropriate candidates for treatment with adalimumab based on
approved local label. [0809] k) Current symptoms of severe,
progressive, or uncontrolled renal, hepatic, hematological,
gastrointestinal, pulmonary, cardiovascular, neurological,
endocrine, metabolic, cutaneous, or psychiatric disease, or any
medical conditions that, in the opinion of the investigator, might
place the subject at unacceptable risk for participation in this
study. [0810] l) Subjects who have a history of known
diverticulitis, perforated diverticular diseases, or small bowel
and/or upper GI perforation. [0811] m) Subjects who have class 3 or
4 congestive heart failure. [0812] n) Subjects who have a history
of any demyelinating disease [0813] o) Subjects who have received a
vaccination with a live vaccine in the 6 weeks before
randomization. Subjects who are in close contact with other people
who have received a live vaccine may be enrolled at the
investigator's discretion. [0814] p) Have present or previous
malignancies, except documented history of cured non metastatic
squamous or basal skin cell carcinoma, or cervical carcinoma in
situ, with no recurrence in the 5 years prior to randomization
[0815] q) Subjects who have undergone a major surgical procedure
within the 60 days prior to randomization. [0816] r) Subjects with
history of surgery on more than 5 joints. [0817] s) Subjects with a
history of (within 12 months of signing the consent), or known
current problems with drug or alcohol abuse or known cirrhosis
including alcoholic cirrhosis. For all subjects, alcohol should not
be consumed within 72 hours prior to study related lab testing, and
subjects should limit their alcohol intake to 4 drink s per week.
[0818] t) Subjects with a history or suspicion of unreliability,
poor cooperation, or non compliance with medical treatment.
3. Physical and Laboratory Test Findings
[0818] [0819] a) Subjects with positive Hepatitis B surface antigen
(HBsAg). If required by local health authorities or medical society
guidelines, subjects at high risk for HBV infection (including
subjects with known family history of HBV infection, latent HBV or
HBV carrier, Hepatitis B surface antibody (HBsAb), personal medical
history of hepatitis or blood transfusion history) must also be
tested for quantitative HBV DNA. Subjects with a positive Hepatitis
B core antibody (HBcAb) must also be tested for quantitative HBV
DNA. Subjects with positive HBsAg or HBV DNA are excluded from the
study. [0820] b) Hepatitis C antibody-positive subjects who are HCV
positive by confirmatory testing, such as by PCR. [0821] c) Have
any clinically significant laboratory abnormalities including but
not limited to: [0822] i. Hepatic [0823] ALT 1.5.times.upper limit
of normal (ULN) [0824] AST 1.5.times.ULN [0825] Total bilirubin
1.5.times.ULN. [0826] ii. Hematologic [0827] Hemoglobin <9 g/dL
[0828] Absolute neutrophil count <1,000/mm3 (1.0.times.109/L)
[0829] Platelets <100,000/mm3 (100.times.109/L) [0830] d) (1 to
3)--.beta.-D glucan positive subjects (only required when, locally,
considered the standard of care)
4. Allergies and Adverse Drug Reaction
[0830] [0831] Subjects who have a known clinically significant
allergy or hypersensitivity to any biologic therapy.
5. Prohibited Therapies
[0831] [0832] a) Subjects who have used the following conventional
synthetic DMARDs less than 4 weeks prior to randomization [0833] i.
chloroquine [0834] ii. hydroxychloroquine [0835] iii. quinacrine
[0836] iv. d-penicillamine [0837] v. azathioprine [0838] vi.
cyclosporine [0839] vii. cyclophosphamide [0840] viii. nimesulide
[0841] ix. tofacitinib [0842] x. Immunoabsorption (ie, Prosorba)
column or cholestyramine [0843] b) Subjects who have used the
following conventional synthetic DMARDs less than 8 weeks prior to
randomization [0844] i. Oral or parenteral gold [0845] ii.
leflunimide [0846] c) Subjects who are undergoing physical therapy
should be on a stable regimen of treatments for 4 weeks prior to
screening. [0847] d) Subjects treated with any biologic DMARD
including, but not limited to: TNF inhibitors, abatacept,
tocilizumab, rituxumab, and investigational biologic therapy.
[0848] e) Subjects treated with IM, IV, or IA corticosteroids less
than 28 days prior to signing informed consent. [0849] f) Subjects
treated with a non-biologic investigational drug within 28 days of
signing informed consent, or less than 5 terminal half lives of its
elimination (whichever is longer). [0850] g) Subjects who are
receiving calcineurin inhibitors at the time of signing informed
consent. [0851] h) Subjects who are receiving nimesulide at the
time of signing informed consent. 6. Other Exclusion Criteria a)
Prisoners or subjects who are involuntarily incarcerated [0852] b)
Subjects who are compulsorily detained for treatment of either a
psychiatric or physical (eg, infectious disease) illness
Corticosteroids for Treatment of RA Symptoms
[0853] All subjects must continue to receive oral prednisone
(<10 mg/day), or its equivalent, at the dose being administered
at the time of signing the informed consent. Intra-vascular (IV),
intra-articular (IA), and intramuscular (IM) corticosteroid
injections are not permitted during the double-blind period.
Analgesics and NSAIDs
[0854] NSAIDs and analgesics (including topical NSAIDs) are not
permitted within 12 hours before a joint evaluation. [0855] NSAIDS
doses should remain stable with the exception of decreases being
permitted due to related adverse events, such as gastric toxicity.
[0856] Analgesics [0857] Acetaminophen (paracetamol) maximal dose 2
g/day with no daily dose exceeding 2.5 g. [0858] NOTE: combination
products including acetaminophen and narcotic analgesics (eg,
acetaminophen with codeine phosphate, acetaminophen with
propoxyphene napsylate, acetaminophen with oxycodone HCl,
acetaminophen with hydrocodone bitartrate, etc.) are allowed
provided the acetaminophen component dosage is accounted for in the
maximum of 2 g/day. [0859] Narcotic analgesics must not exceed 30
mg/day of morphine or its equivalent and are not permitted within
12 hours before a joint evaluation. [0860] Tramadol, gabapentin,
and pregabalin are allowed but doses must be stable throughout the
double-blind period. [0861] Acetylsalicylic acid is allowed in low
doses (eg, 100 mg/day) for cardiovascular prophylaxis Herbal
medications and Dietary Supplements
[0862] The subject anti-IL-6 antibody optionally may be used in
combination with specific herbal medications or supplements.
However, the following herbal medications and dietary supplements
that have potential hepatotoxic effects and ideally but not
necessarily should be avoided: [0863] Ba Jiao Lian [0864] Cascara
[0865] Chaparral [0866] Chi R Yun [0867] Comfrey [0868] Ephedra
[0869] Flavocoxid [0870] Germander [0871] Greater Celandine [0872]
Green Tea extracts [0873] Jin Bu Huan [0874] Kava Kava [0875]
Margosa Oil [0876] Ma Huang [0877] Pennyroyal Oil [0878] Senna
(high dose or long term use) [0879] Sho Saiko To and Dai Saiko To
[0880] Shou Wu Pian [0881] Usnic Acid
Prohibited Treatments
Prohibited Treatments During Double-Blind Period
[0881] [0882] Conventional synthetic DMARDs other than methotrexate
(including but not limited to sulfasalazine, hydroxychloroquine,
chloroquine) [0883] adrenal corticotropic hormone (ACTH) [0884]
chloroquine [0885] hydroxychloroquine [0886] oral or parenteral
gold [0887] quinacrine [0888] d-penicillamine [0889] leflunomide
[0890] azathioprine [0891] cyclosporine [0892] nimesulide [0893]
All investigational and approved biologic RA therapies other than
clazakizumab (including but not limited to abatacept, tocilizumab,
etanercept, anakinra, infliximab, rituximab, etc) [0894] Use of any
investigational drug other than study medication [0895]
Intra-articular injections of hyaluronic acid [0896]
Immunoabsorption (ie, Prosorba) column or cholestyramine
Prohibited Treatments During Long Term Extension (LTE)
[0897] Prohibited treatments during the LTE are the same as during
Double-Blind Period except for the following: [0898] Sulfasalazine
at labeled doses for rheumatoid arthritis is permitted during this
period [0899] Intra-articular injections of hyaluronic acid may be
given during this period however must be limited to 1 or 2 joints,
and are permitted one time every six months. Intra-articular
steroid injections and intra-articular hyaluronic acid injections
must not occur within the same 6 month period.
Other Restrictions and Precautions
[0900] The prescribing label of all concomitant medications used as
subject's background therapy should be evaluated by the
investigator for continued administration during the subject's
participation in this study (eg, known toxicities, drug-drug
interactions).
Discontinuation of Subjects from Treatment:
[0901] Subjects MUST discontinue investigational product (and
non-investigational product at the discretion of the investigator)
for any of the following reasons: [0902] Use of prohibited
medication [0903] Pregnancy [0904] Missed Doses [0905] Missed more
than one dose of investigational product for any reason during the
first 12 weeks of the double-blind period [0906] Missed more than
two consecutive doses of investigational product for any reason
after the first 12 weeks of the double-blind period [0907] Any
clinical adverse event (AE), laboratory abnormality or intercurrent
illness which, in the opinion of the investigator, indicates that
continued participation in the study is not in the best interest of
the subject (eg, significant LFT abnormalities, GI perforation
etc.) [0908] Severe liver enzyme elevations. [0909] Positive
testing for TB. [0910] Subject's request to stop study treatment or
withdrawal of informed consent [0911] Unblinding a subject for any
reason (emergency or non-emergency) [0912] Loss of ability to
freely provide consent through imprisonment or involuntarily
incarceration for treatment of either a psychiatric or physical
(eg, infectious disease) illness
[0913] If study treatment is discontinued prior to the subject's
completion of the study, the reason for the discontinuation must be
documented in the subject's medical records and entered on the
appropriate case report form (CRF) page.
Withdrawal of Consent
[0914] Subjects who request to discontinue study treatment will
remain in the study and must continue to be followed for protocol
specified follow-up procedures. The only exception to this is when
a subject specifically withdraws consent for any further contact
with him/her or persons previously authorized by subject to provide
this information. Subjects should notify the investigator of the
decision to withdraw consent from future follow-up in writing,
whenever possible. The withdrawal of consent should be explained in
detail in the medical records by the investigator, as to whether
the withdrawal is from further treatment with study drug only or
also from study procedures and/or post treatment study follow-up,
and entered on the appropriate CRF page. In the event that vital
status (whether the subject is alive or dead) is being measured,
publicly available information should be used to determine vital
status only as appropriately directed in accordance with local
law.
Treatments:
[0915] A pharmaceutical form of an active substance or placebo is
used as a reference in a clinical study, including products already
with a marketing authorization but used or assembled (formulated or
packaged) in a way different from the authorized form, or used for
an unauthorized indication, or when used to gain further
information about the authorized form.
[0916] In this protocol, investigational product(s) is/are: [0917]
Clazakizumab SC injection, 25 mg/syringe (25 mg/ml) [0918]
Clazakizumab Placebo, SC injection, 0.9% Sodium chloride [0919]
Adalimumab (Humira.RTM.) SC injection, 40 mg/pre-filled syringe (40
mg/0.8 ml) [0920] Adalimumab Placebo, SC injection pre-filled
syringe, 0.9% Sodium chloride, 0.8 ml/syringe [0921] Methotrexate,
2.5 mg tablet
[0922] A "double-dummy" design is used to protect the blind during
the double-blind period. Depending on randomization assignment,
subjects will receive the following regimens: Weeks 0-11: [0923]
Treatment Arm 1: one SC injection of clazakizumab every 4 weeks
PLUS one SC injection of adalimumab placebo every 2 weeks [0924]
Treatment Arm 2: one SC injection of clazakizumab placebo (D5W)
every 4 weeks PLUS one SC injection of adalimumab every 2 weeks
[0925] Treatment Arm 3: one SC injection of clazakizumab placebo
(D5W) every 4 weeks PLUS one SC injection of adalimumab placebo
every 2 weeks Weeks 12-24 (including week 24 visit): [0926]
Treatment Arm 1: one SC injection of clazakizumab every 4 weeks
PLUS one SC injection of adalimumab placebo every 2 weeks [0927]
Treatment Arm 2: one SC injection of clazakizumab every 4 weeks
PLUS one SC injection of adalimumab every 2 weeks [0928] Treatment
Arm 3: one SC injection of clazakizumab placebo (D5W) every 4 weeks
PLUS one SC injection of adalimumab placebo every 2 weeks
Week 28 (LTE Week 4):
[0928] [0929] Treatment Arm 1: one SC injection of clazakizumab
[0930] Treatment Arm 2: one SC injection of clazakizumab [0931]
Treatment Arm 3: one SC injection of clazakizumab placebo (D5W)
Week 32 (LTE Week 8) and duration of LTE (open-label clazakizumab):
[0932] Treatment Arm 1: one SC injection of clazakizumab [0933]
Treatment Arm 2: one SC injection of clazakizumab [0934] Treatment
Arm 3: one SC injection of clazakizumab
[0935] Injections sites may include the upper arms, thigh, or
abdomen. It is recommended that no more than one injection should
occur per injection site at a given visit. At each visit,
corresponding sites on both sides should be used. When possible,
injection sites should be rotated between visits. MTX is maintained
at the same dose as at randomization (rounded to the nearest 2.5 mg
increment).
Outcomes Research Assessments
[0936] During and after treatment pain physical functioning,
disease, fatigue assessments will be performed. Based on the
results obtained in RA and PsA patients to date these clinical
trials will provide further evidence that the subject anti-IL-6
antibodies are safe and effective at low dosages for treating
rheumatoid arthritis as well as psoriatic arthritis and moreover
provide for greater patient remission and at lower dosages
administered less frequently than current biologics used to treat
RA and that these low dosages are further effective in RA patients
resistant to DMARDS such as e.g., MTX.
[0937] Also, these clinical trials will provide further evidence
that the subject anti-IL-6 antibodies are safe and effective at low
dosages and that they may be self-administered such as by the use
of an auto-injector pen thus simplifying patient compliance and
better allowing rheumatoid or psoriatic arthritis patients to
manage their disease.
[0938] Although the subject technology has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications will practiced within the scope of the appended
claims. Modifications of the above-described modes for carrying out
the subject technology that are obvious to persons of skill in
medicine, pharmacology, microbiology, and/or related fields are
intended to be within the scope of the following claims.
[0939] All publications (e.g., Non-Patent Literature), patent
application publications, and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this subject technology pertains. All such
publications (e.g., Non-Patent Literature), patent application
publications, and patent applications are herein incorporated by
reference to the same extent as if each individual publication,
patent, patent application publication, or patent application is
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
7481183PRTArtificialSynthetic Polypeptide 1Val Pro Pro Gly Glu Asp
Ser Lys Asp Val Ala Ala Pro His Arg Gln 1 5 10 15 Pro Leu Thr Ser
Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu 20 25 30 Asp Gly
Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met 35 40 45
Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro 50
55 60 Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu
Glu 65 70 75 80 Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe
Glu Val Tyr 85 90 95 Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser
Glu Glu Gln Ala Arg 100 105 110 Ala Val Gln Met Ser Thr Lys Val Leu
Ile Gln Phe Leu Gln Lys Lys 115 120 125 Ala Lys Asn Leu Asp Ala Ile
Thr Thr Pro Asp Pro Thr Thr Asn Ala 130 135 140 Ser Leu Leu Thr Lys
Leu Gln Ala Gln Asn Gln Trp Leu Gln Asp Met 145 150 155 160 Thr Thr
His Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser 165 170 175
Leu Arg Ala Leu Arg Gln Met 180 2163PRTArtificialSynthetic
Polypeptide 2Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr
Gln Thr Pro Ala Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val
Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln Ser Ile Asn Asn Glu Leu
Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Arg Pro Lys Leu Leu
Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Ser Ser Arg
Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile
Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105
110 Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu
115 120 125 Val Val Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu 145 150 155 160 Leu Asn Asn
3166PRTArtificialSynthetic Polypeptide 3Met Glu Thr Gly Leu Arg Trp
Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser
Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro
Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40 45 Asn
Tyr Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55
60 Trp Ile Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp
65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val
Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr
Tyr Phe Cys Ala 100 105 110 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys
Leu Val Lys 165 411PRTArtificialSynthetic Peptide 4Gln Ala Ser Gln
Ser Ile Asn Asn Glu Leu Ser 1 5 10 57PRTArtificialSynthetic Peptide
5Arg Ala Ser Thr Leu Ala Ser 1 5 612PRTArtificialSynthetic Peptide
6Gln Gln Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala 1 5 10
75PRTArtificialSynthetic Peptide 7Asn Tyr Tyr Val Thr 1 5
816PRTArtificialSynthetic Peptide 8Ile Ile Tyr Gly Ser Asp Glu Thr
Ala Tyr Ala Thr Trp Ala Ile Gly 1 5 10 15 912PRTArtificialSynthetic
Peptide 9Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 1 5 10
10491DNAArtificialSynthetic Sequence 10atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac
ccagactcca gcctcggtgt ctgcagctgt gggaggcaca 120gtcaccatca
agtgccaggc cagtcagagc attaacaatg aattatcctg gtatcagcag
180aaaccagggc agcgtcccaa gctcctgatc tatagggcat ccactctggc
atctggggtc 240tcatcgcggt tcaaaggcag tggatctggg acagagttca
ctctcaccat cagcgacctg 300gagtgtgccg atgctgccac ttactactgt
caacagggtt atagtctgag gaatattgat 360aatgctttcg gcggagggac
cgaggtggtg gtcaaacgta cggtagcggc cccatctgtc 420ttcatcttcc
cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg
480ctgaataact t 49111499DNAArtificialSynthetic Sequence
11atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag
60tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac actcacctgc
120acagcctctg gattctccct cagtaactac tacgtgacct gggtccgcca
ggctccaggg 180aaggggctgg aatggatcgg aatcatttat ggtagtgatg
aaacggccta cgcgacctgg 240gcgataggcc gattcaccat ctccaaaacc
tcgaccacgg tggatctgaa aatgaccagt 300ctgacagccg cggacacggc
cacctatttc tgtgccagag atgatagtag tgactgggat 360gcaaaattta
acttgtgggg ccaaggcacc ctggtcaccg tctcgagcgc ctccaccaag
420ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg
cacagcggcc 480ctgggctgcc tggtcaagg 4991233DNAArtificialSynthetic
Sequence 12caggccagtc agagcattaa caatgaatta tcc
331321DNAArtificialSynthetic Sequence 13agggcatcca ctctggcatc t
211436DNAArtificialSynthetic Sequence 14caacagggtt atagtctgag
gaatattgat aatgct 361515DNAArtificialSynthetic Sequence
15aactactacg tgacc 151648DNAArtificialSynthetic Sequence
16atcatttatg gtagtgatga aacggcctac gcgacctggg cgataggc
481736DNAArtificialSynthetic Sequence 17gatgatagta gtgactggga
tgcaaaattt aacttg 3618109PRTArtificialSynthetic Polypeptide 18Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr
20 25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala
Thr Trp Ala Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp
Trp Asp Ala Lys Phe Asn Leu 100 105 19109PRTArtificialSynthetic
Polypeptide 19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Leu Ser Asn Tyr 20 25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp
Glu Thr Ala Tyr Ala Thr Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 100 105
2099PRTArtificialSynthetic Polypeptide 20Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15 Arg Val Thr Ile
Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu Leu 20 25 30 Ser Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45
Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50
55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu
Arg Asn Ile 85 90 95 Asp Asn Ala 21170PRTArtificialSynthetic
Polypeptide 21Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr
Gln Thr Pro Ala Ser 20 25 30 Val Glu Val Ala Val Gly Gly Thr Val
Thr Ile Asn Cys Gln Ala Ser 35 40 45 Glu Thr Ile Tyr Ser Trp Leu
Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu
Ile Tyr Gln Ala Ser Asp Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg
Phe Ser Gly Ser Gly Ala Gly Thr Glu Tyr Thr Leu Thr 85 90 95 Ile
Ser Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105
110 Gly Tyr Ser Gly Ser Asn Val Asp Asn Val Phe Gly Gly Gly Thr Glu
115 120 125 Val Val Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala
Lys 165 170 22167PRTArtificialSynthetic Polypeptide 22Met Glu Thr
Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val
Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Arg Leu Val Thr 20 25
30 Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu
35 40 45 Asn Asp His Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60 Glu Tyr Ile Gly Phe Ile Asn Ser Gly Gly Ser Ala
Arg Tyr Ala Ser 65 70 75 80 Trp Ala Glu Gly Arg Phe Thr Ile Ser Arg
Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Leu Thr Thr
Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Val Arg Gly Gly Ala Val
Trp Ser Ile His Ser Phe Asp Pro Trp Gly 115 120 125 Pro Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155
160 Ala Leu Gly Cys Leu Val Lys 165 2311PRTArtificialSynthetic
Peptide 23Gln Ala Ser Glu Thr Ile Tyr Ser Trp Leu Ser 1 5 10
247PRTArtificialSynthetic Peptide 24Gln Ala Ser Asp Leu Ala Ser 1 5
2512PRTArtificialSynthetic Peptide 25Gln Gln Gly Tyr Ser Gly Ser
Asn Val Asp Asn Val 1 5 10 265PRTArtificialSynthetic Peptide 26Asp
His Ala Met Gly 1 5 2716PRTArtificialSynthetic Peptide 27Phe Ile
Asn Ser Gly Gly Ser Ala Arg Tyr Ala Ser Trp Ala Glu Gly 1 5 10 15
2812PRTArtificialSynthetic Peptide 28Gly Gly Ala Val Trp Ser Ile
His Ser Phe Asp Pro 1 5 10 29511DNAArtificialSynthetic Sequence
29atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60agatgtgcct atgatatgac ccagactcca gcctctgtgg aggtagctgt gggaggcaca
120gtcaccatca attgccaggc cagtgagacc atttacagtt ggttatcctg
gtatcagcag 180aagccagggc agcctcccaa gctcctgatc taccaggcat
ccgatctggc atctggggtc 240ccatcgcgat tcagcggcag tggggctggg
acagagtaca ctctcaccat cagcggcgtg 300cagtgtgacg atgctgccac
ttactactgt caacagggtt atagtggtag taatgttgat 360aatgttttcg
gcggagggac cgaggtggtg gtcaaacgta cggtagcggc cccatctgtc
420ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt
tgtgtgcctg 480ctgaataact tctatcccag agaggccaaa g
51130501DNAArtificialSynthetic Sequence 30atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60gagcagctga aggagtccgg
gggtcgcctg gtcacgcctg ggacacccct gacacttacc 120tgcacagcct
ctggattctc cctcaatgac catgcaatgg gctgggtccg ccaggctcca
180gggaaggggc tggaatacat cggattcatt aatagtggtg gtagcgcacg
ctacgcgagc 240tgggcagaag gccgattcac catctccaga acctcgacca
cggtggatct gaaaatgacc 300agtctgacaa ccgaggacac ggccacctat
ttctgtgtca gagggggtgc tgtttggagt 360attcatagtt ttgatccctg
gggcccaggg accctggtca ccgtctcgag cgcctccacc 420aagggcccat
cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg
480gccctgggct gcctggtcaa g 5013133DNAArtificialSynthetic Sequence
31caggccagtg agaccattta cagttggtta tcc 333221DNAArtificialSynthetic
Sequence 32caggcatccg atctggcatc t 213336DNAArtificialSynthetic
Sequence 33caacagggtt atagtggtag taatgttgat aatgtt
363415DNAArtificialSynthetic Sequence 34gaccatgcaa tgggc
153548DNAArtificialSynthetic Sequence 35ttcattaata gtggtggtag
cgcacgctac gcgagctggg cagaaggc 483636DNAArtificialSynthetic
Sequence 36gggggtgctg tttggagtat tcatagtttt gatccc
3637165PRTArtificialSynthetic Polypeptide 37Met Asp Thr Arg Ala Pro
Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala
Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser
Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser 35 40 45
Gln Ser Val Tyr Asp Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro 50
55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu Ala
Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Val Gly Ser Gly Ser Gly Thr
Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val Gln Cys Asp Asp Ala
Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Tyr Asp Asp Asp Ser Asp
Asn Ala Phe Gly Gly Gly Thr 115 120 125 Glu Val Val Val Lys Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140 Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu
Asn Asn Phe 165 38166PRTArtificialSynthetic Polypeptide 38Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20
25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu
Ser 35 40 45 Val Tyr Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Phe Ile Thr Met Ser Asp Asn Ile
Asn Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys
Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr Thr
Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Ser Arg Gly Trp
Gly Thr Met Gly Arg Leu Asp Leu Trp Gly Pro 115 120 125 Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150
155 160 Leu Gly Cys Leu Val Lys 165 3913PRTArtificialSynthetic
Peptide 39Gln Ala Ser Gln Ser Val Tyr Asp Asn Asn Tyr Leu Ser 1 5
10 407PRTArtificialSynthetic Peptide 40Gly Ala Ser Thr Leu Ala Ser
1
5 4111PRTArtificialSynthetic Peptide 41Ala Gly Val Tyr Asp Asp Asp
Ser Asp Asn Ala 1 5 10 425PRTArtificialSynthetic Peptide 42Val Tyr
Tyr Met Asn 1 5 4316PRTArtificialSynthetic Peptide 43Phe Ile Thr
Met Ser Asp Asn Ile Asn Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
4412PRTArtificialSynthetic Peptide 44Ser Arg Gly Trp Gly Thr Met
Gly Arg Leu Asp Leu 1 5 10 45496DNAArtificialSynthetic Sequence
45atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60acatttgccg ccgtgctgac ccagactcca tctcccgtgt ctgcagctgt gggaggcaca
120gtcagcatca gttgccaggc cagtcagagt gtttatgaca acaactactt
atcctggttt 180cagcagaaac cagggcagcc tcccaagctc ctgatctatg
gtgcatccac tctggcatct 240ggggtcccat cgcggttcgt gggcagtgga
tctgggacac agttcactct caccatcaca 300gacgtgcagt gtgacgatgc
tgccacttac tattgtgcag gcgtttatga tgatgatagt 360gataatgcct
tcggcggagg gaccgaggtg gtggtcaaac gtacggtagc ggccccatct
420gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc
tgttgtgtgc 480ctgctgaata acttct 49646499DNAArtificialSynthetic
Sequence 46atggagactg ggctgcgctg gcttctcctg gtggctgtgc tcaaaggtgt
ccagtgtcag 60tcgctggagg agtccggggg tcgcctggtc acccctggga cacccctgac
actcacctgc 120acagcctctg gattctccct cagtgtctac tacatgaact
gggtccgcca ggctccaggg 180aaggggctgg aatggatcgg attcattaca
atgagtgata atataaatta cgcgagctgg 240gcgaaaggcc gattcaccat
ctccaaaacc tcgaccacgg tggatctgaa aatgaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgccagga gtcgtggctg gggtacaatg
360ggtcggttgg atctctgggg cccaggcacc ctcgtcaccg tctcgagcgc
ctccaccaag 420ggcccatcgg tcttccccct ggcaccctcc tccaagagca
cctctggggg cacagcggcc 480ctgggctgcc tggtcaagg
4994739DNAArtificialSynthetic Sequence 47caggccagtc agagtgttta
tgacaacaac tacttatcc 394821DNAArtificialSynthetic Sequence
48ggtgcatcca ctctggcatc t 214933DNAArtificialSynthetic Sequence
49gcaggcgttt atgatgatga tagtgataat gcc 335015DNAArtificialSynthetic
Sequence 50gtctactaca tgaac 155148DNAArtificialSynthetic Sequence
51ttcattacaa tgagtgataa tataaattac gcgagctggg cgaaaggc
485236DNAArtificialSynthetic Sequence 52agtcgtggct ggggtacaat
gggtcggttg gatctc 3653164PRTArtificialSynthetic Polypeptide 53Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Ile Cys Asp Pro Val Leu Thr Gln Thr Pro Ser Pro
20 25 30 Val Ser Ala Pro Val Gly Gly Thr Val Ser Ile Ser Cys Gln
Ala Ser 35 40 45 Gln Ser Val Tyr Glu Asn Asn Tyr Leu Ser Trp Phe
Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly
Ala Ser Thr Leu Asp Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Tyr
Asp Asp Asp Ser Asp Asp Ala Phe Gly Gly Gly Thr 115 120 125 Glu Val
Val Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145
150 155 160 Leu Leu Asn Asn 54167PRTArtificialSynthetic Polypeptide
54Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1
5 10 15 Val Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Gly Leu Val
Thr 20 25 30 Pro Gly Gly Thr Leu Thr Leu Thr Cys Thr Ala Ser Gly
Phe Ser Leu 35 40 45 Asn Ala Tyr Tyr Met Asn Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Phe Ile Thr Leu Asn
Asn Asn Val Ala Tyr Ala Asn 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr
Phe Ser Lys Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser
Pro Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Ser
Arg Gly Trp Gly Ala Met Gly Arg Leu Asp Leu Trp Gly 115 120 125 His
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135
140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
145 150 155 160 Ala Leu Gly Cys Leu Val Lys 165
5513PRTArtificialSynthetic Peptide 55Gln Ala Ser Gln Ser Val Tyr
Glu Asn Asn Tyr Leu Ser 1 5 10 567PRTArtificialSynthetic Peptide
56Gly Ala Ser Thr Leu Asp Ser 1 5 5711PRTArtificialSynthetic
Peptide 57Ala Gly Val Tyr Asp Asp Asp Ser Asp Asp Ala 1 5 10
585PRTArtificialSynthetic Peptide 58Ala Tyr Tyr Met Asn 1 5
5916PRTArtificialSynthetic Peptide 59Phe Ile Thr Leu Asn Asn Asn
Val Ala Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15
6012PRTArtificialSynthetic Peptide 60Ser Arg Gly Trp Gly Ala Met
Gly Arg Leu Asp Leu 1 5 10 61494DNAArtificialSynthetic Sequence
61atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc
60atatgtgacc ctgtgctgac ccagactcca tctcccgtat ctgcacctgt gggaggcaca
120gtcagcatca gttgccaggc cagtcagagt gtttatgaga acaactattt
atcctggttt 180cagcagaaac cagggcagcc tcccaagctc ctgatctatg
gtgcatccac tctggattct 240ggggtcccat cgcggttcaa aggcagtgga
tctgggacac agttcactct caccattaca 300gacgtgcagt gtgacgatgc
tgccacttac tattgtgcag gcgtttatga tgatgatagt 360gatgatgcct
tcggcggagg gaccgaggtg gtggtcaaac gtacggtagc ggccccatct
420gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc
tgttgtgtgc 480ctgctgaata actt 49462502DNAArtificialSynthetic
Sequence 62atggagactg ggctgcgctg gcttctcctg gtggctgtgc tcaaaggtgt
ccagtgtcag 60gagcagctga aggagtccgg aggaggcctg gtaacgcctg gaggaaccct
gacactcacc 120tgcacagcct ctggattctc cctcaatgcc tactacatga
actgggtccg ccaggctcca 180gggaaggggc tggaatggat cggattcatt
actctgaata ataatgtagc ttacgcgaac 240tgggcgaaag gccgattcac
cttctccaaa acctcgacca cggtggatct gaaaatgacc 300agtccgacac
ccgaggacac ggccacctat ttctgtgcca ggagtcgtgg ctggggtgca
360atgggtcggt tggatctctg gggccatggc accctggtca ccgtctcgag
cgcctccacc 420aagggcccat cggtcttccc cctggcaccc tcctccaaga
gcacctctgg gggcacagcg 480gccctgggct gcctggtcaa gg
5026339DNAArtificialSynthetic Sequence 63caggccagtc agagtgttta
tgagaacaac tatttatcc 396421DNAArtificialSynthetic Sequence
64ggtgcatcca ctctggattc t 216533DNAArtificialSynthetic Sequence
65gcaggcgttt atgatgatga tagtgatgat gcc 336615DNAArtificialSynthetic
Sequence 66gcctactaca tgaac 156748DNAArtificialSynthetic Sequence
67ttcattactc tgaataataa tgtagcttac gcgaactggg cgaaaggc
486836DNAArtificialSynthetic Sequence 68agtcgtggct ggggtgcaat
gggtcggttg gatctc 3669164PRTArtificialSynthetic Polypeptide 69Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Pro
20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln
Ala Ser 35 40 45 Gln Ser Val Asp Asp Asn Asn Trp Leu Gly Trp Tyr
Gln Gln Lys Arg 50 55 60 Gly Gln Pro Pro Lys Tyr Leu Ile Tyr Ser
Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Leu
Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Gly Phe
Ser Gly Asn Ile Phe Ala Phe Gly Gly Gly Thr Glu 115 120 125 Val Val
Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145
150 155 160 Leu Asn Asn Phe 70164PRTArtificialSynthetic Polypeptide
70Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1
5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr
Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe
Ser Leu Ser 35 40 45 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Gly Gly Phe Gly
Thr Thr Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Arg Ile Thr Ser Pro
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Gly Gly
Pro Gly Asn Gly Gly Asp Ile Trp Gly Gln Gly Thr Leu 115 120 125 Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 130 135
140 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
145 150 155 160 Leu Val Lys Asp 7113PRTArtificialSynthetic Peptide
71Gln Ala Ser Gln Ser Val Asp Asp Asn Asn Trp Leu Gly 1 5 10
727PRTArtificialSynthetic Peptide 72Ser Ala Ser Thr Leu Ala Ser 1 5
7310PRTArtificialSynthetic Peptide 73Ala Gly Gly Phe Ser Gly Asn
Ile Phe Ala 1 5 10 745PRTArtificialSynthetic Peptide 74Ser Tyr Ala
Met Ser 1 5 7516PRTArtificialSynthetic Peptide 75Ile Ile Gly Gly
Phe Gly Thr Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
769PRTArtificialSynthetic Peptide 76Gly Gly Pro Gly Asn Gly Gly Asp
Ile 1 5 77493DNAArtificialSynthetic Sequence 77atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgccc
aagtgctgac ccagactcca tcgcctgtgt ctgcagctgt gggaggcaca
120gtcaccatca actgccaggc cagtcagagt gttgatgata acaactggtt
aggctggtat 180cagcagaaac gagggcagcc tcccaagtac ctgatctatt
ctgcatccac tctggcatct 240ggggtcccat cgcggttcaa aggcagtgga
tctgggacac agttcactct caccatcagc 300gacctggagt gtgacgatgc
tgccacttac tactgtgcag gcggttttag tggtaatatc 360tttgctttcg
gcggagggac cgaggtggtg gtcaaacgta cggtagcggc cccatctgtc
420ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt
tgtgtgcctg 480ctgaataact tct 49378493DNAArtificialSynthetic
Sequence 78atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcggtggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagtctctg gcttctccct cagtagctat gcaatgagct
gggtccgcca ggctccagga 180aaggggctgg agtggatcgg aatcattggt
ggttttggta ccacatacta cgcgacctgg 240gcgaaaggcc gattcaccat
ctccaaaacc tcgaccacgg tggatctgag aatcaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgccagag gtggtcctgg taatggtggt
360gacatctggg gccaagggac cctggtcacc gtctcgagcg cctccaccaa
gggcccatcg 420gtcttccccc tggcaccctc ctccaagagc acctctgggg
gcacagcggc cctgggctgc 480ctggtcaagg act
4937939DNAArtificialSynthetic Sequence 79caggccagtc agagtgttga
tgataacaac tggttaggc 398021DNAArtificialSynthetic Sequence
80tctgcatcca ctctggcatc t 218130DNAArtificialSynthetic Sequence
81gcaggcggtt ttagtggtaa tatctttgct 308215DNAArtificialSynthetic
Sequence 82agctatgcaa tgagc 158348DNAArtificialSynthetic Sequence
83atcattggtg gttttggtac cacatactac gcgacctggg cgaaaggc
488427DNAArtificialSynthetic Sequence 84ggtggtcctg gtaatggtgg
tgacatc 2785164PRTArtificialSynthetic Polypeptide 85Met Asp Thr Arg
Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro
Gly Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30
Val Ser Val Pro Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ser Ser 35
40 45 Gln Ser Val Tyr Asn Asn Phe Leu Ser Trp Tyr Gln Gln Lys Pro
Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu
Ala Ser Gly 65 70 75 80 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Gln Phe Thr Leu 85 90 95 Thr Ile Ser Gly Val Gln Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys Leu 100 105 110 Gly Gly Tyr Asp Asp Asp Ala
Asp Asn Ala Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160
Leu Asn Asn Phe 86170PRTArtificialSynthetic Polypeptide 86Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20
25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu
Ser 35 40 45 Asp Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Ala Gly Ser Gly Ser
Thr Trp Tyr Ala Ser 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile Ser
Lys Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Ile Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Asp Gly Tyr
Asp Asp Tyr Gly Asp Phe Asp Arg Leu Asp Leu 115 120 125 Trp Gly Pro
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150
155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170
8712PRTArtificialSynthetic Peptide 87Gln Ser Ser Gln Ser Val Tyr
Asn Asn Phe Leu Ser 1 5 10 887PRTArtificialSynthetic Peptide 88Gln
Ala Ser Lys Leu Ala Ser 1 5 8911PRTArtificialSynthetic Peptide
89Leu Gly Gly Tyr Asp Asp Asp Ala Asp Asn Ala 1 5 10
905PRTArtificialSynthetic Peptide 90Asp Tyr Ala Met Ser 1 5
9117PRTArtificialSynthetic Peptide 91Ile Ile Tyr Ala Gly Ser Gly
Ser Thr Trp Tyr Ala Ser Trp Ala Lys 1 5 10 15 Gly
9214PRTArtificialSynthetic Peptide 92Asp Gly Tyr Asp Asp Tyr Gly
Asp Phe Asp Arg Leu Asp Leu 1 5 10 93492DNAArtificialSynthetic
Sequence 93atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgcag ccgtgctgac ccagacacca tcgcccgtgt ctgtacctgt
gggaggcaca 120gtcaccatca agtgccagtc cagtcagagt gtttataata
atttcttatc gtggtatcag 180cagaaaccag ggcagcctcc caagctcctg
atctaccagg catccaaact ggcatctggg 240gtcccagata ggttcagcgg
cagtggatct gggacacagt tcactctcac catcagcggc 300gtgcagtgtg
acgatgctgc cacttactac tgtctaggcg gttatgatga tgatgctgat
360aatgctttcg gcggagggac cgaggtggtg gtcaaacgta cggtagcggc
cccatctgtc 420ttcatcttcc cgccatctga tgagcagttg aaatctggaa
ctgcctctgt tgtgtgcctg 480ctgaataact tc
49294511DNAArtificialSynthetic Sequence 94atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac gctcacctgc 120acagtctctg
gaatcgacct cagtgactat gcaatgagct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatcatttat gctggtagtg gtagcacatg
gtacgcgagc 240tgggcgaaag gccgattcac
catctccaaa acctcgacca cggtggatct gaaaatcacc 300agtccgacaa
ccgaggacac ggccacctat ttctgtgcca gagatggata cgatgactat
360ggtgatttcg atcgattgga tctctggggc ccaggcaccc tcgtcaccgt
ctcgagcgcc 420tccaccaagg gcccatcggt cttccccctg gcaccctcct
ccaagagcac ctctgggggc 480acagcggccc tgggctgcct ggtcaaggac t
5119536DNAArtificialSynthetic Sequence 95cagtccagtc agagtgttta
taataatttc ttatcg 369621DNAArtificialSynthetic Sequence
96caggcatcca aactggcatc t 219733DNAArtificialArtificial Sequence
97ctaggcggtt atgatgatga tgctgataat gct 339815DNAArtificialSynthetic
Sequence 98gactatgcaa tgagc 159951DNAArtificialSynthetic Sequence
99atcatttatg ctggtagtgg tagcacatgg tacgcgagct gggcgaaagg c
5110042DNAArtificialSynthetic Sequence 100gatggatacg atgactatgg
tgatttcgat cgattggatc tc 42101164PRTArtificialSynthetic Polypeptide
101Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro
Ala Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys
Cys Gln Ala Ser 35 40 45 Gln Ser Ile Asn Asn Glu Leu Ser Trp Tyr
Gln Gln Lys Ser Gly Gln 50 55 60 Arg Pro Lys Leu Leu Ile Tyr Arg
Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu
Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr
Ser Leu Arg Asn Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu 115 120 125
Val Val Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130
135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu 145 150 155 160 Leu Asn Asn Phe 102166PRTArtificialSynthetic
Polypeptide 102Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Ser Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Ala Ser Gly Phe Ser Leu Ser 35 40 45 Asn Tyr Tyr Met Thr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Met Ile
Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Asn Trp 65 70 75 80 Ala Ile Gly
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys
Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln
115 120 125 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 130 135 140 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 145 150 155 160 Leu Gly Cys Leu Val Lys 165
10311PRTArtificialSynthetic Peptide 103Gln Ala Ser Gln Ser Ile Asn
Asn Glu Leu Ser 1 5 10 1047PRTArtificialSynthetic Peptide 104Arg
Ala Ser Thr Leu Ala Ser 1 5 10512PRTArtificialSynthetic Peptide
105Gln Gln Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala 1 5 10
1065PRTArtificialSynthetic Peptide 106Asn Tyr Tyr Met Thr 1 5
10716PRTArtificialSynthetic Peptide 107Met Ile Tyr Gly Ser Asp Glu
Thr Ala Tyr Ala Asn Trp Ala Ile Gly 1 5 10 15
10812PRTArtificialSynthetic Peptide 108Asp Asp Ser Ser Asp Trp Asp
Ala Lys Phe Asn Leu 1 5 10 109492DNAArtificialSynthetic Sequence
109atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct atgatatgac ccagactcca gcctcggtgt ctgcagctgt
gggaggcaca 120gtcaccatca aatgccaggc cagtcagagc attaacaatg
aattatcctg gtatcagcag 180aaatcagggc agcgtcccaa gctcctgatc
tatagggcat ccactctggc atctggggtc 240tcatcgcggt tcaaaggcag
tggatctggg acagagttca ctctcaccat cagcgacctg 300gagtgtgccg
atgctgccac ttactactgt caacagggtt atagtctgag gaatattgat
360aatgctttcg gcggagggac cgaggtggtg gtcaaacgta cggtagcggc
cccatctgtc 420ttcatcttcc cgccatctga tgagcagttg aaatctggaa
ctgcctctgt tgtgtgcctg 480ctgaataact tc
492110499DNAArtificialSynthetic Sequence 110atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tctcaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtaactac tacatgacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatgatttat ggtagtgatg aaacagccta
cgcgaactgg 240gcgataggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ctgacagccg cggacacggc cacctatttc
tgtgccagag atgatagtag tgactgggat 360gcaaaattta acttgtgggg
ccaagggacc ctcgtcaccg tctcgagcgc ctccaccaag 420ggcccatcgg
tcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc
480ctgggctgcc tggtcaagg 49911133DNAArtificialSynthetic Sequence
111caggccagtc agagcattaa caatgaatta tcc
3311221DNAArtificialSynthetic Sequence 112agggcatcca ctctggcatc t
2111336DNAArtificialSynthetic Sequence 113caacagggtt atagtctgag
gaatattgat aatgct 3611415DNAArtificialSynthetic Sequence
114aactactaca tgacc 1511548DNAArtificialSynthetic Sequence
115atgatttatg gtagtgatga aacagcctac gcgaactggg cgataggc
4811636DNAArtificialSynthetic Sequence 116gatgatagta gtgactggga
tgcaaaattt aacttg 36117109PRTArtificialSynthetic Polypeptide 117Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr
20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Gly Met Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala
Asn Trp Ala Ile 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp
Trp Asp Ala Lys Phe Asn Leu 100 105 118109PRTArtificialSynthetic
Polypeptide 118Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Leu Ser Asn Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Met Ile Tyr Gly Ser Asp
Glu Thr Ala Tyr Ala Asn Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 100 105
119100PRTArtificialSynthetic Polypeptide 119Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu Ser
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser
Leu Arg Asn 85 90 95 Ile Asp Asn Ala 100
12016PRTArtificialSynthetic Peptide 120Ile Ile Tyr Gly Ser Asp Glu
Thr Ala Tyr Ala Thr Ser Ala Ile Gly 1 5 10 15
12116PRTArtificialSynthetic Peptide 121Met Ile Tyr Gly Ser Asp Glu
Thr Ala Tyr Ala Asn Ser Ala Ile Gly 1 5 10 15
122123PRTArtificialSynthetic Polypeptide 122Met Asp Thr Arg Ala Pro
Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala
Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val Ser
Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ser Ser 35 40 45
Gln Ser Val Gly Asn Asn Gln Asp Leu Ser Trp Phe Gln Gln Arg Pro 50
55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Glu Ile Ser Lys Leu Glu
Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
His Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp Ala
Ala Thr Tyr Tyr Cys 100 105 110 Leu Gly Gly Tyr Asp Asp Asp Ala Asp
Asn Ala 115 120 123128PRTArtificialSynthetic Polypeptide 123Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys His Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20
25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
Ser 35 40 45 Ser Arg Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Tyr Ile Trp Ser Gly Gly Ser Thr
Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys
Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr
Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Leu Gly Asp Thr
Gly Gly His Ala Tyr Ala Thr Arg Leu Asn Leu 115 120 125
12413PRTArtificialSynthetic Peptide 124Gln Ser Ser Gln Ser Val Gly
Asn Asn Gln Asp Leu Ser 1 5 10 1257PRTArtificialSynthetic Peptide
125Glu Ile Ser Lys Leu Glu Ser 1 5 12611PRTArtificialSynthetic
Peptide 126Leu Gly Gly Tyr Asp Asp Asp Ala Asp Asn Ala 1 5 10
1275PRTArtificialSynthetic Peptide 127Ser Arg Thr Met Ser 1 5
12816PRTArtificialSynthetic Peptide 128Tyr Ile Trp Ser Gly Gly Ser
Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
12915PRTArtificialSynthetic Peptide 129Leu Gly Asp Thr Gly Gly His
Ala Tyr Ala Thr Arg Leu Asn Leu 1 5 10 15
130369DNAArtificialSynthetic Sequence 130atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgcag ccgtgctgac
ccagacacca tcacccgtgt ctgcagctgt gggaggcaca 120gtcaccatca
gttgccagtc cagtcagagt gttggtaata accaggactt atcctggttt
180cagcagagac cagggcagcc tcccaagctc ctgatctacg aaatatccaa
actggaatct 240ggggtcccat cgcggttcag cggcagtgga tctgggacac
acttcactct caccatcagc 300ggcgtacagt gtgacgatgc tgccacttac
tactgtctag gcggttatga tgatgatgct 360gataatgct
369131384DNAArtificialSynthetic Sequence 131atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcac 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cagtagtcgt acaatgtcct gggtccgcca ggctccaggg
180aaggggctgg agtggatcgg atacatttgg agtggtggta gcacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagat tgggcgatac tggtggtcac 360gcttatgcta ctcgcttaaa tctc
38413239DNAArtificialSynthetic Sequence 132cagtccagtc agagtgttgg
taataaccag gacttatcc 3913321DNAArtificialSynthetic Sequence
133gaaatatcca aactggaatc t 2113433DNAArtificialSynthetic Sequence
134ctaggcggtt atgatgatga tgctgataat gct
3313515DNAArtificialSynthetic Sequence 135agtcgtacaa tgtcc
1513648DNAArtificialSynthetic Sequence 136tacatttgga gtggtggtag
cacatactac gcgacctggg cgaaaggc 4813745DNAArtificialSynthetic
Sequence 137ttgggcgata ctggtggtca cgcttatgct actcgcttaa atctc
45138123PRTArtificialSynthetic Polypeptide 138Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Ser 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ser Ser 35 40
45 Gln Ser Val Tyr Ser Asn Lys Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Thr Ser Lys Leu
Ala Ser 65 70 75 80 Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Leu Gly Ala Tyr Asp Asp Asp Ala
Asp Asn Ala 115 120 139126PRTArtificialSynthetic Polypeptide 139Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Lys Pro
20 25 30 Asp Glu Thr Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
Leu Glu 35 40 45 Gly Gly Tyr Met Thr Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ser Tyr Asp Ser Gly Ser
Thr Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser
Lys Thr Ser Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Leu
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Val Arg Ser Leu
Lys Tyr Pro Thr Val Thr Ser Asp Asp Leu 115 120 125
14013PRTArtificialSynthetic Peptide 140Gln Ser Ser Gln Ser Val Tyr
Ser Asn Lys Tyr Leu Ala 1 5 10 1417PRTArtificialSynthetic Peptide
141Trp Thr Ser Lys Leu Ala Ser 1 5 14211PRTArtificialSynthetic
Peptide 142Leu Gly Ala Tyr Asp Asp Asp Ala Asp Asn Ala 1 5 10
1435PRTArtificialSynthetic Peptide 143Gly Gly Tyr Met Thr 1 5
14416PRTArtificialSynthetic Peptide 144Ile Ser Tyr Asp Ser Gly Ser
Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
14512PRTArtificialSynthetic Peptide 145Ser Leu Lys Tyr Pro Thr Val
Thr Ser Asp Asp Leu 1 5 10 146369DNAArtificialSynthetic Sequence
146atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgcag ccgtgctgac ccagacacca tcgtccgtgt ctgcagctgt
gggaggcaca 120gtcagcatca gttgccagtc cagtcagagt gtttatagta
ataagtacct agcctggtat 180cagcagaaac cagggcagcc tcccaagctc
ctgatctact ggacatccaa actggcatct 240ggggccccat cacggttcag
cggcagtgga tctgggacac aattcactct caccatcagc 300ggcgtgcagt
gtgacgatgc tgccacttac tactgtctag gcgcttatga tgatgatgct 360gataatgct
369147378DNAArtificialSynthetic Sequence 147atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggaag agtccggggg
tcgcctggtc aagcctgacg aaaccctgac actcacctgc 120acagcctctg
gattctccct ggagggcggc tacatgacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg
aatcagttat gatagtggta gcacatacta cgcgagctgg 240gcgaaaggcc
gattcaccat ctccaagacc tcgtcgacca cggtggatct gaaaatgacc
300agtctgacaa ccgaggacac ggccacctat ttctgcgtca gatcactaaa
atatcctact 360gttacttctg atgacttg 37814839DNAArtificialSynthetic
Sequence 148cagtccagtc agagtgttta tagtaataag tacctagcc
3914921DNAArtificialSynthetic Sequence 149tggacatcca aactggcatc t
2115033DNAArtificialSynthetic Sequence 150ctaggcgctt atgatgatga
tgctgataat gct 3315115DNAArtificialSynthetic Sequence 151ggcggctaca
tgacc 1515248DNAArtificialSynthetic Sequence 152atcagttatg
atagtggtag cacatactac gcgagctggg cgaaaggc
4815336DNAArtificialSynthetic Sequence 153tcactaaaat atcctactgt
tacttctgat gacttg 36154123PRTArtificialSynthetic Polypeptide 154Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro
20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln
Ser Ser 35 40 45 Gln Ser Val Tyr Asn Asn Asn Asp Leu Ala Trp Tyr
Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Tyr
Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val
Gln Cys Asp Asp Ala Ala Ala Tyr Tyr Cys 100 105 110 Leu Gly Gly Tyr
Asp Asp Asp Ala Asp Asn Ala 115 120 155129PRTArtificialSynthetic
Polypeptide 155Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Leu Ser Leu Ser 35 40 45 Ser Asn Thr Ile Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Tyr Ile
Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Ser Trp 65 70 75 80 Val Asn Gly
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys
Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Gly Gly Tyr Ala Ser Gly Gly Tyr Pro Tyr Ala Thr Arg Leu Asp
115 120 125 Leu 15613PRTArtificialSynthetic Peptide 156Gln Ser Ser
Gln Ser Val Tyr Asn Asn Asn Asp Leu Ala 1 5 10
1577PRTArtificialSynthetic Peptide 157Tyr Ala Ser Thr Leu Ala Ser 1
5 15811PRTArtificialSynthetic Peptide 158Leu Gly Gly Tyr Asp Asp
Asp Ala Asp Asn Ala 1 5 10 1595PRTArtificialSynthetic Peptide
159Ser Asn Thr Ile Asn 1 5 16016PRTArtificialSynthetic Peptide
160Tyr Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Val Asn Gly
1 5 10 15 16116PRTArtificialSynthetic Peptide 161Gly Gly Tyr Ala
Ser Gly Gly Tyr Pro Tyr Ala Thr Arg Leu Asp Leu 1 5 10 15
162369DNAArtificialSynthetic Sequence 162atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgcag ccgtgctgac
ccagacacca tcacccgtgt ctgcagctgt gggaggcaca 120gtcaccatca
gttgccagtc cagtcagagt gtttataata ataacgactt agcctggtat
180cagcagaaac cagggcagcc tcctaaactc ctgatctatt atgcatccac
tctggcatct 240ggggtcccat cgcggttcaa aggcagtgga tctgggacac
agttcactct caccatcagc 300ggcgtgcagt gtgacgatgc tgccgcttac
tactgtctag gcggttatga tgatgatgct 360gataatgct
369163387DNAArtificialSynthetic Sequence 163atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtatctg
gattatccct cagtagcaat acaataaact gggtccgcca ggctccaggg
180aaggggctgg agtggatcgg atacatttgg agtggtggta gtacatacta
cgcgagctgg 240gtgaatggtc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag ggggttacgc tagtggtggt 360tatccttatg ccactcggtt ggatctc
38716439DNAArtificialSynthetic Sequence 164cagtccagtc agagtgttta
taataataac gacttagcc 3916521DNAArtificialSynthetic Sequence
165tatgcatcca ctctggcatc t 2116633DNAArtificialSynthetic Sequence
166ctaggcggtt atgatgatga tgctgataat gct
3316715DNAArtificialSynthetic Sequence 167agcaatacaa taaac
1516848DNAArtificialSynthetic Sequence 168tacatttgga gtggtggtag
tacatactac gcgagctggg tgaatggt 4816948DNAArtificialSynthetic
Sequence 169gggggttacg ctagtggtgg ttatccttat gccactcggt tggatctc
48170123PRTArtificialSynthetic Polypeptide 170Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Ser 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser 35 40
45 Gln Ser Val Tyr Asn Asn Asp Tyr Leu Ser Trp Tyr Gln Gln Arg Pro
50 55 60 Gly Gln Arg Pro Lys Leu Leu Ile Tyr Gly Ala Ser Lys Leu
Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly
Lys Gln Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val Gln Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Leu Gly Asp Tyr Asp Asp Asp Ala
Asp Asn Thr 115 120 171123PRTArtificialSynthetic Polypeptide 171Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Thr
Leu Ser 35 40 45 Thr Asn Tyr Tyr Leu Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Ile Ile Tyr Pro Ser Gly
Asn Thr Tyr Cys Ala Lys 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Ser Thr Thr Val 85 90 95 Asp Leu Lys Met Thr Ser
Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe 100 105 110 Cys Ala Arg Asn
Tyr Gly Gly Asp Glu Ser Leu 115 120 17213PRTArtificialSynthetic
Peptide 172Gln Ser Ser Gln Ser Val Tyr Asn Asn Asp Tyr Leu Ser 1 5
10 1737PRTArtificialSynthetic Peptide 173Gly Ala Ser Lys Leu Ala
Ser 1 5 17411PRTArtificialSynthetic Peptide 174Leu Gly Asp Tyr Asp
Asp Asp Ala Asp Asn Thr 1 5 10 1756PRTArtificialSynthetic Peptide
175Thr Asn Tyr Tyr Leu Ser 1 5 17616PRTArtificialSynthetic Peptide
176Ile Ile Tyr Pro Ser Gly Asn Thr Tyr Cys Ala Lys Trp Ala Lys Gly
1 5 10 15 1778PRTArtificialSynthetic Peptide 177Asn Tyr Gly Gly Asp
Glu Ser Leu 1 5 178369DNAArtificialSynthetic Sequence 178atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgcag
ccgtgctgac ccagacacca tcctccgtgt ctgcagctgt gggaggcaca
120gtcaccatca attgccagtc cagtcagagt gtttataata acgactactt
atcctggtat 180caacagaggc cagggcaacg tcccaagctc ctaatctatg
gtgcttccaa actggcatct 240ggggtcccgt cacggttcaa aggcagtgga
tctgggaaac agtttactct caccatcagc 300ggcgtgcagt gtgacgatgc
tgccacttac tactgtctgg gcgattatga tgatgatgct 360gataatact
369179369DNAArtificialSynthetic Sequence 179atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacttgc 120acagtctctg
gattcaccct cagtaccaac tactacctga gctgggtccg ccaggctcca
180gggaaggggc tagaatggat cggaatcatt tatcctagtg gtaacacata
ttgcgcgaag 240tgggcgaaag gccgattcac catctccaaa acctcgtcga
ccacggtgga tctgaaaatg 300accagtccga caaccgagga cacagccacg
tatttctgtg ccagaaatta tggtggtgat 360gaaagtttg
36918039DNAArtificialSynthetic Sequence 180cagtccagtc agagtgttta
taataacgac tacttatcc 3918121DNAArtificialSynthetic Sequence
181ggtgcttcca aactggcatc t 2118233DNAArtificialSynthetic Sequence
182ctgggcgatt atgatgatga tgctgataat act
3318318DNAArtificialSynthetic Sequence 183accaactact acctgagc
1818448DNAArtificialSynthetic Sequence 184atcatttatc ctagtggtaa
cacatattgc gcgaagtggg cgaaaggc 4818524DNAArtificialSynthetic
Sequence 185aattatggtg gtgatgaaag tttg
24186119PRTArtificialSynthetic Polypeptide 186Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser 20 25 30 Val
Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40
45 Glu Thr Ile Gly Asn Ala Leu Ala Trp Tyr Gln Gln Lys Ser Gly Gln
50 55 60 Pro Pro Lys Leu Leu Ile Tyr Lys Ala Ser Lys Leu Ala Ser
Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu
Tyr Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala
Thr Tyr Tyr Cys Gln Trp 100 105 110 Cys Tyr Phe Gly Asp Ser Val 115
187128PRTArtificialSynthetic Polypeptide 187Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Thr Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Glu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Glu
Gly Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Asp Phe 35 40 45
Ser Ser Gly Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50
55 60 Leu Glu Trp Ile Ala Cys Ile Phe Thr Ile Thr Thr Asn Thr Tyr
Tyr 65 70 75 80 Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr
Ser Ser Thr 85 90 95 Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala
Ala Asp Thr Ala Thr 100 105 110 Tyr Leu Cys Ala Arg Gly Ile Tyr Ser
Asp Asn Asn Tyr Tyr Ala Leu 115 120 125 18811PRTArtificialSynthetic
Peptide 188Gln Ala Ser Glu Thr Ile Gly Asn Ala Leu Ala 1 5 10
1897PRTArtificialSynthetic Peptide 189Lys Ala Ser Lys Leu Ala Ser 1
5 1909PRTArtificialSynthetic Peptide 190Gln Trp Cys Tyr Phe Gly Asp
Ser Val 1 5 1916PRTArtificialSynthetic Peptide 191Ser Gly Tyr Tyr
Met Cys 1 5 19217PRTArtificialSynthetic Peptide 192Cys Ile Phe Thr
Ile Thr Thr Asn Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 Gly
19311PRTArtificialSynthetic Peptide 193Gly Ile Tyr Ser Asp Asn Asn
Tyr Tyr Ala Leu 1 5 10 194357DNAArtificialSynthetic Sequence
194atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgatg ttgtgatgac ccagactcca gcctccgtgg aggcagctgt
gggaggcaca 120gtcaccatca agtgccaggc cagtgagacc attggcaatg
cattagcctg gtatcagcag 180aaatcagggc agcctcccaa gctcctgatc
tacaaggcat ccaaactggc atctggggtc 240ccatcgcggt tcaaaggcag
tggatctggg acagagtaca ctctcaccat cagcgacctg 300gagtgtgccg
atgctgccac ttactactgt caatggtgtt attttggtga tagtgtt
357195384DNAArtificialSynthetic Sequence 195atggagactg ggctgcgctg
gcttctcctg gtcactgtgc tcaaaggtgt ccagtgtcag 60gagcagctgg tggagtccgg
gggaggcctg gtccagcctg agggatccct gacactcacc 120tgcacagcct
ctggattcga cttcagtagc ggctactaca tgtgctgggt ccgccaggct
180ccagggaagg ggctggagtg gatcgcgtgt attttcacta ttactactaa
cacttactac 240gcgagctggg cgaaaggccg attcaccatc tccaagacct
cgtcgaccac ggtgactctg 300caaatgacca gtctgacagc cgcggacacg
gccacctatc tctgtgcgag agggatttat 360tctgataata attattatgc cttg
38419633DNAArtificialSynthetic Sequence 196caggccagtg agaccattgg
caatgcatta gcc 3319721DNAArtificialSynthetic Sequence 197aaggcatcca
aactggcatc t 2119827DNAArtificialSynthetic Sequence 198caatggtgtt
attttggtga tagtgtt 2719918DNAArtificialSynthetic Sequence
199agcggctact acatgtgc 1820051DNAArtificialSynthetic Sequence
200tgtattttca ctattactac taacacttac tacgcgagct gggcgaaagg c
5120133DNAArtificialSynthetic Sequence 201gggatttatt ctgataataa
ttattatgcc ttg 33202119PRTArtificialSynthetic Polypeptide 202Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser
20 25 30 Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln
Ala Ser 35 40 45 Glu Ser Ile Gly Asn Ala Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile Tyr Lys Ala Ser
Thr Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Gly Val Gln Cys
Ala Asp Ala Ala Ala Tyr Tyr Cys Gln Trp 100 105 110 Cys Tyr Phe Gly
Asp Ser Val 115 203128PRTArtificialSynthetic Polypeptide 203Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys Gln Gln Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20
25 30 Pro Gly Ala Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Ser
Phe 35 40 45 Ser Ser Gly Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro
Gly Lys Gly 50 55 60 Leu Glu Ser Ile Ala Cys Ile Phe Thr Ile Thr
Asp Asn Thr Tyr Tyr 65 70 75 80 Ala Asn Trp Ala Lys Gly Arg Phe Thr
Ile Ser Lys Pro Ser Ser Pro 85 90 95 Thr Val Thr Leu Gln Met Thr
Ser Leu Thr Ala Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg
Gly Ile Tyr Ser Thr Asp Asn Tyr Tyr Ala Leu 115 120 125
20411PRTArtificialSynthetic Peptide 204Gln Ala Ser Glu Ser Ile Gly
Asn Ala Leu Ala 1 5 10 2057PRTArtificialSynthetic Peptide 205Lys
Ala Ser Thr Leu Ala Ser 1 5 2069PRTArtificialSynthetic Peptide
206Gln Trp Cys Tyr Phe Gly Asp Ser Val 1 5
2076PRTArtificialSynthetic Peptide 207Ser Gly Tyr Tyr Met Cys 1 5
20817PRTArtificialSynthetic Peptide 208Cys Ile Phe Thr Ile Thr Asp
Asn Thr Tyr Tyr Ala Asn Trp Ala Lys 1 5 10 15 Gly
20911PRTArtificialSynthetic Peptide 209Gly Ile Tyr Ser Thr Asp Asn
Tyr Tyr Ala Leu 1 5 10 210357DNAArtificialSynthetic Sequence
210atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgatg ttgtgatgac ccagactcca gcctccgtgg aggcagctgt
gggaggcaca 120gtcaccatca agtgccaggc
cagtgagagc attggcaatg cattagcctg gtatcagcag 180aaaccagggc
agcctcccaa gctcctgatc tacaaggcat ccactctggc atctggggtc
240ccatcgcggt tcagcggcag tggatctggg acagagttca ctctcaccat
cagcggcgtg 300cagtgtgccg atgctgccgc ttactactgt caatggtgtt
attttggtga tagtgtt 357211384DNAArtificialSynthetic Sequence
211atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60cagcagctgg tggagtccgg gggaggcctg gtcaagccgg gggcatccct
gacactcacc 120tgcaaagcct ctggattctc cttcagtagc ggctactaca
tgtgctgggt ccgccaggct 180ccagggaagg ggctggagtc gatcgcatgc
atttttacta ttactgataa cacttactac 240gcgaactggg cgaaaggccg
attcaccatc tccaagccct cgtcgcccac ggtgactctg 300caaatgacca
gtctgacagc cgcggacacg gccacctatt tctgtgcgag ggggatttat
360tctactgata attattatgc cttg 38421233DNAArtificialSynthetic
Sequence 212caggccagtg agagcattgg caatgcatta gcc
3321321DNAArtificialSynthetic Sequence 213aaggcatcca ctctggcatc t
2121427DNAArtificialSynthetic Sequence 214caatggtgtt attttggtga
tagtgtt 2721518DNAArtificialSynthetic Sequence 215agcggctact
acatgtgc 1821651DNAArtificialSynthetic Sequence 216tgcattttta
ctattactga taacacttac tacgcgaact gggcgaaagg c
5121733DNAArtificialSynthetic Sequence 217gggatttatt ctactgataa
ttattatgcc ttg 33218123PRTArtificialSynthetic Polypeptide 218Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser
20 25 30 Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln
Ala Ser 35 40 45 Gln Ser Val Ser Ser Tyr Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser
Thr Leu Glu Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys 100 105 110 Thr Tyr Gly Thr
Ser Ser Ser Tyr Gly Ala Ala 115 120 219133PRTArtificialSynthetic
Polypeptide 219Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Ile Ser Leu Ser 35 40 45 Ser Asn Ala Ile Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile
Ser Tyr Ser Gly Thr Thr Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly
Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp 85 90 95 Leu
Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105
110 Ala Arg Asp Asp Pro Thr Thr Val Met Val Met Leu Ile Pro Phe Gly
115 120 125 Ala Gly Met Asp Leu 130 22011PRTArtificialSynthetic
Peptide 220Gln Ala Ser Gln Ser Val Ser Ser Tyr Leu Asn 1 5 10
2217PRTArtificialSynthetic Peptide 221Arg Ala Ser Thr Leu Glu Ser 1
5 22213PRTArtificialSynthetic Peptide 222Gln Cys Thr Tyr Gly Thr
Ser Ser Ser Tyr Gly Ala Ala 1 5 10 2235PRTArtificialSynthetic
Peptide 223Ser Asn Ala Ile Ser 1 5 22416PRTArtificialSynthetic
Peptide 224Ile Ile Ser Tyr Ser Gly Thr Thr Tyr Tyr Ala Ser Trp Ala
Lys Gly 1 5 10 15 22519PRTArtificialSynthetic Peptide 225Asp Asp
Pro Thr Thr Val Met Val Met Leu Ile Pro Phe Gly Ala Gly 1 5 10 15
Met Asp Leu 226369DNAArtificialSynthetic Sequence 226atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgatg
ttgtgatgac ccagactcca gcctccgtgg aggcagctgt gggaggcaca
120gtcaccatca agtgccaggc cagtcagagc gttagtagct acttaaactg
gtatcagcag 180aaaccagggc agcctcccaa gctcctgatc tacagggcat
ccactctgga atctggggtc 240ccatcgcggt tcaaaggcag tggatctggg
acagagttca ctctcaccat cagcgacctg 300gagtgtgccg atgctgccac
ttactactgt caatgtactt atggtactag tagtagttat 360ggtgctgct
369227399DNAArtificialSynthetic Sequence 227atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120accgtctctg
gtatctccct cagtagcaat gcaataagct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatcattagt tatagtggta ccacatacta
cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgtcgacca
cggtggatct gaaaatcact 300agtccgacaa ccgaggacac ggccacctac
ttctgtgcca gagatgaccc tacgacagtt 360atggttatgt tgataccttt
tggagccggc atggacctc 39922833DNAArtificialSynthetic Sequence
228caggccagtc agagcgttag tagctactta aac
3322921DNAArtificialSynthetic Sequence 229agggcatcca ctctggaatc t
2123039DNAArtificialSynthetic Sequence 230caatgtactt atggtactag
tagtagttat ggtgctgct 3923115DNAArtificialSynthetic Sequence
231agcaatgcaa taagc 1523248DNAArtificialSynthetic Sequence
232atcattagtt atagtggtac cacatactac gcgagctggg cgaaaggc
4823357DNAArtificialSynthetic Sequence 233gatgacccta cgacagttat
ggttatgttg ataccttttg gagccggcat ggacctc
57234125PRTArtificialSynthetic Polypeptide 234Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Ala Ser Pro 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40
45 Gln Ser Val Tyr Lys Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro
50 55 60 Gly Gln Pro Pro Lys Gly Leu Ile Tyr Ser Ala Ser Thr Leu
Asp Ser 65 70 75 80 Gly Val Pro Leu Arg Phe Ser Gly Ser Gly Ser Gly
Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Val Gln Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Leu Gly Ser Tyr Asp Cys Ser Ser
Gly Asp Cys Tyr Ala 115 120 125 235119PRTArtificialSynthetic
Polypeptide 235Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly
Asp Leu Val Lys Pro 20 25 30 Glu Gly Ser Leu Thr Leu Thr Cys Thr
Ala Ser Gly Phe Ser Phe Ser 35 40 45 Ser Tyr Trp Met Cys Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Ala Cys Ile
Val Thr Gly Asn Gly Asn Thr Tyr Tyr Ala Asn 65 70 75 80 Trp Ala Lys
Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val 85 90 95 Thr
Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe 100 105
110 Cys Ala Lys Ala Tyr Asp Leu 115 23613PRTArtificialSynthetic
Peptide 236Gln Ala Ser Gln Ser Val Tyr Lys Asn Asn Tyr Leu Ser 1 5
10 2377PRTArtificialSynthetic Peptide 237Ser Ala Ser Thr Leu Asp
Ser 1 5 23813PRTArtificialSynthetic Peptide 238Leu Gly Ser Tyr Asp
Cys Ser Ser Gly Asp Cys Tyr Ala 1 5 10 2395PRTArtificialSynthetic
Peptide 239Ser Tyr Trp Met Cys 1 5 24017PRTArtificialSynthetic
Peptide 240Cys Ile Val Thr Gly Asn Gly Asn Thr Tyr Tyr Ala Asn Trp
Ala Lys 1 5 10 15 Gly 2414PRTArtificialSynthetic Peptide 241Ala Tyr
Asp Leu 1 242375DNAArtificialSynthetic Sequence 242atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgccc
aagtgctgac ccagactgca tcgcccgtgt ctgcagctgt gggaggcaca
120gtcaccatca actgccaggc cagtcagagt gtttataaga acaactactt
atcctggtat 180cagcagaaac cagggcagcc tcccaaaggc ctgatctatt
ctgcatcgac tctagattct 240ggggtcccat tgcggttcag cggcagtgga
tctgggacac agttcactct caccatcagc 300gacgtgcagt gtgacgatgc
tgccacttac tactgtctag gcagttatga ttgtagtagt 360ggtgattgtt atgct
375243357DNAArtificialSynthetic Sequence 243atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgttggagg agtccggggg
agacctggtc aagcctgagg gatccctgac actcacctgc 120acagcctctg
gattctcctt cagtagctac tggatgtgct gggtccgcca ggctccaggg
180aaggggctgg agtggatcgc atgcattgtt actggtaatg gtaacactta
ctacgcgaac 240tgggcgaaag gccgattcac catctccaaa acctcgtcga
ccacggtgac tctgcaaatg 300accagtctga cagccgcgga cacggccacc
tatttttgtg cgaaagccta tgacttg 35724439DNAArtificialSynthetic
Sequence 244caggccagtc agagtgttta taagaacaac tacttatcc
3924521DNAArtificialSynthetic Sequence 245tctgcatcga ctctagattc t
2124639DNAArtificialSynthetic Sequence 246ctaggcagtt atgattgtag
tagtggtgat tgttatgct 3924715DNAArtificialSynthetic Sequence
247agctactgga tgtgc 1524851DNAArtificialSynthetic Sequence
248tgcattgtta ctggtaatgg taacacttac tacgcgaact gggcgaaagg c
5124912DNAArtificialSynthetic Sequence 249gcctatgact tg
12250123PRTArtificialSynthetic Polypeptide 250Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ser Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser 35 40
45 Gln Ser Val Tyr Asp Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro
50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu
Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Thr Gly Ser Gly
Thr Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val Gln Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Phe Asn Asp Asp Ser
Asp Asp Ala 115 120 251125PRTArtificialSynthetic Polypeptide 251Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Pro Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Leu Ser Gly Phe Ser
Leu Ser 35 40 45 Ala Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu 50 55 60 Trp Ile Gly Phe Ile Thr Leu Ser Asp His
Ile Ser Tyr Ala Arg Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser
Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Ser Arg Gly
Trp Gly Ala Met Gly Arg Leu Asp Leu 115 120 125
25213PRTArtificialSynthetic Peptide 252Gln Ala Ser Gln Ser Val Tyr
Asp Asn Asn Tyr Leu Ser 1 5 10 2537PRTArtificialSynthetic Peptide
253Gly Ala Ser Thr Leu Ala Ser 1 5 25411PRTArtificialSynthetic
Peptide 254Ala Gly Val Phe Asn Asp Asp Ser Asp Asp Ala 1 5 10
2555PRTArtificialSynthetic Peptide 255Ala Tyr Tyr Met Ser 1 5
25616PRTArtificialSynthetic Peptide 256Phe Ile Thr Leu Ser Asp His
Ile Ser Tyr Ala Arg Trp Ala Lys Gly 1 5 10 15
25712PRTArtificialSynthetic Peptide 257Ser Arg Gly Trp Gly Ala Met
Gly Arg Leu Asp Leu 1 5 10 258369DNAArtificialSynthetic Sequence
258atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggttcc 60acatttgccg ccgtgctgac ccagactcca tctcccgtgt ctgcagctgt
gggaggcaca 120gtcagcatca gttgccaggc cagtcagagt gtttatgaca
acaactattt atcctggtat 180cagcagaaac caggacagcc tcccaagctc
ctgatctatg gtgcatccac tctggcatct 240ggggtcccat cgcggttcaa
aggcacggga tctgggacac agttcactct caccatcaca 300gacgtgcagt
gtgacgatgc tgccacttac tattgtgcag gcgtttttaa tgatgatagt 360gatgatgcc
369259375DNAArtificialSynthetic Sequence 259atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc ccaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acactctctg
gattctccct cagtgcatac tatatgagct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg attcattact ctgagtgatc atatatctta
cgcgaggtgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagga gtcgtggctg gggtgcaatg 360ggtcggttgg atctc
37526039DNAArtificialSynthetic Sequence 260caggccagtc agagtgttta
tgacaacaac tatttatcc 3926121DNAArtificialSynthetic Sequence
261ggtgcatcca ctctggcatc t 2126233DNAArtificialSynthetic Sequence
262gcaggcgttt ttaatgatga tagtgatgat gcc
3326315DNAArtificialSynthetic Sequence 263gcatactata tgagc
1526448DNAArtificialSynthetic Sequence 264ttcattactc tgagtgatca
tatatcttac gcgaggtggg cgaaaggc 4826536DNAArtificialSynthetic
Sequence 265agtcgtggct ggggtgcaat gggtcggttg gatctc
36266123PRTArtificialSynthetic Polypeptide 266Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Thr Ile Ser Cys Gln Ala Ser 35 40
45 Gln Ser Val Tyr Asn Asn Lys Asn Leu Ala Trp Tyr Gln Gln Lys Ser
50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Leu
Ala Ser 65 70 75 80 Gly Val Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Gln Phe Thr 85 90 95 Leu Thr Val Ser Gly Val Gln Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Leu Gly Val Phe Asp Asp Asp Ala
Asp Asn Ala 115 120 267121PRTArtificialSynthetic Polypeptide 267Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
Leu Ser 35 40 45 Ser Tyr Ser Met Thr Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu 50 55 60 Tyr Ile Gly Val Ile Gly Thr Ser Gly Ser
Thr Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser
Arg Thr Ser Thr Thr Val Ala Leu 85 90 95 Lys Ile Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Val 100 105 110 Arg Ser Leu Ser
Ser Ile Thr Phe Leu 115 120 26813PRTArtificialSynthetic Peptide
268Gln Ala Ser Gln Ser Val Tyr Asn Asn Lys Asn Leu Ala 1 5 10
2697PRTArtificialSynthetic Peptide 269Trp Ala Ser Thr Leu Ala Ser 1
5 27011PRTArtificialSynthetic Peptide 270Leu Gly Val Phe Asp Asp
Asp Ala Asp Asn Ala 1 5 10
2715PRTArtificialSynthetic Peptide 271Ser Tyr Ser Met Thr 1 5
27216PRTArtificialSynthetic Peptide 272Val Ile Gly Thr Ser Gly Ser
Thr Tyr Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
2738PRTArtificialSynthetic Peptide 273Ser Leu Ser Ser Ile Thr Phe
Leu 1 5 274369DNAArtificialSynthetic Sequence 274atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acattcgcag
ccgtgctgac ccagacacca tcgcccgtgt ctgcggctgt gggaggcaca
120gtcaccatca gttgccaggc cagtcagagt gtttataaca acaaaaattt
agcctggtat 180cagcagaaat cagggcagcc tcccaagctc ctgatctact
gggcatccac tctggcatct 240ggggtctcat cgcggttcag cggcagtgga
tctgggacac agttcactct caccgtcagc 300ggcgtgcagt gtgacgatgc
tgccacttac tactgtctag gcgtttttga tgatgatgct 360gataatgct
369275363DNAArtificialSynthetic Sequence 275atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccaatgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtagctac tccatgacct gggtccgcca ggctccaggg
180aaggggctgg aatatatcgg agtcattggt actagtggta gcacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctccagaacc tcgaccacgg
tggctctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgtcagga gtctttcttc tattactttc 360ttg
36327639DNAArtificialSynthetic Sequence 276caggccagtc agagtgttta
taacaacaaa aatttagcc 3927721DNAArtificialSynthetic Sequence
277tgggcatcca ctctggcatc t 2127833DNAArtificialSynthetic Sequence
278ctaggcgttt ttgatgatga tgctgataat gct
3327915DNAArtificialSynthetic Sequence 279agctactcca tgacc
1528048DNAArtificialSynthetic Sequence 280gtcattggta ctagtggtag
cacatactac gcgacctggg cgaaaggc 4828124DNAArtificialSynthetic
Sequence 281agtctttctt ctattacttt cttg
24282120PRTArtificialSynthetic Polypeptide 282Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys Ala Phe Glu Leu Thr Gln Thr Pro Ala Ser 20 25 30 Val
Glu Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40
45 Gln Asn Ile Tyr Arg Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60 Pro Pro Lys Phe Leu Ile Tyr Leu Ala Ser Thr Leu Ala Ser
Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala
Thr Tyr Tyr Cys Gln Ser 100 105 110 Tyr Tyr Ser Ser Asn Ser Val Ala
115 120 283128PRTArtificialSynthetic Polypeptide 283Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Glu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln 20 25 30
Pro Glu Gly Ser Leu Thr Leu Thr Cys Thr Ala Ser Glu Leu Asp Phe 35
40 45 Ser Ser Gly Tyr Trp Ile Cys Trp Val Arg Gln Val Pro Gly Lys
Gly 50 55 60 Leu Glu Trp Ile Gly Cys Ile Tyr Thr Gly Ser Ser Gly
Ser Thr Phe 65 70 75 80 Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile
Ser Lys Thr Ser Ser 85 90 95 Thr Thr Val Thr Leu Gln Met Thr Ser
Leu Thr Ala Ala Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Ala Arg Gly
Tyr Ser Gly Phe Gly Tyr Phe Lys Leu 115 120 125
28411PRTArtificialSynthetic Peptide 284Gln Ala Ser Gln Asn Ile Tyr
Arg Tyr Leu Ala 1 5 10 2857PRTArtificialSynthetic Peptide 285Leu
Ala Ser Thr Leu Ala Ser 1 5 28610PRTArtificialSynthetic Peptide
286Gln Ser Tyr Tyr Ser Ser Asn Ser Val Ala 1 5 10
2876PRTArtificialSynthetic Peptide 287Ser Gly Tyr Trp Ile Cys 1 5
28818PRTArtificialSynthetic Peptide 288Cys Ile Tyr Thr Gly Ser Ser
Gly Ser Thr Phe Tyr Ala Ser Trp Ala 1 5 10 15 Lys Gly
28910PRTArtificialSynthetic Peptide 289Gly Tyr Ser Gly Phe Gly Tyr
Phe Lys Leu 1 5 10 290360DNAArtificialSynthetic Sequence
290atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcat tcgaattgac ccagactcca gcctccgtgg aggcagctgt
gggaggcaca 120gtcaccatca attgccaggc cagtcagaac atttatagat
acttagcctg gtatcagcag 180aaaccagggc agcctcccaa gttcctgatc
tatctggcat ctactctggc atctggggtc 240ccatcgcggt ttaaaggcag
tggatctggg acagagttca ctctcaccat cagcgacctg 300gagtgtgccg
atgctgccac ttactactgt caaagttatt atagtagtaa tagtgtcgct
360291384DNAArtificialSynthetic Sequence 291atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60gagcagctgg tggagtccgg
gggagacctg gtccagcctg agggatccct gacactcacc 120tgcacagctt
ctgagttaga cttcagtagc ggctactgga tatgctgggt ccgccaggtt
180ccagggaagg ggctggagtg gatcggatgc atttatactg gtagtagtgg
tagcactttt 240tacgcgagtt gggcgaaagg ccgattcacc atctccaaaa
cctcgtcgac cacggtgact 300ctgcaaatga ccagtctgac agccgcggac
acggccacct atttctgtgc gagaggttat 360agtggctttg gttactttaa gttg
38429233DNAArtificialSynthetic Sequence 292caggccagtc agaacattta
tagatactta gcc 3329321DNAArtificialSynthetic Sequence 293ctggcatcta
ctctggcatc t 2129430DNAArtificialSynthetic Sequence 294caaagttatt
atagtagtaa tagtgtcgct 3029518DNAArtificialSynthetic Sequence
295agcggctact ggatatgc 1829654DNAArtificialSynthetic Sequence
296tgcatttata ctggtagtag tggtagcact ttttacgcga gttgggcgaa aggc
5429730DNAArtificialSynthetic Sequence 297ggttatagtg gctttggtta
ctttaagttg 30298122PRTArtificialSynthetic Polypeptide 298Met Asp
Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20
25 30 Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala
Ser 35 40 45 Glu Asp Ile Tyr Arg Leu Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile Tyr Asp Ser Ser Asp
Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser
Gly Thr Glu Phe Thr Leu Ala 85 90 95 Ile Ser Gly Val Gln Cys Asp
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Ala Trp Ser Tyr Ser
Asp Ile Asp Asn Ala 115 120 299123PRTArtificialSynthetic
Polypeptide 299Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Ala Ser Gly Phe Ser Leu Ser 35 40 45 Ser Tyr Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile
Thr Thr Ser Gly Asn Thr Phe Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly
Arg Leu Thr Ile Ser Arg Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys
Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Thr Ser Asp Ile Phe Tyr Tyr Arg Asn Leu 115 120
30011PRTArtificialSynthetic Peptide 300Gln Ala Ser Glu Asp Ile Tyr
Arg Leu Leu Ala 1 5 10 3017PRTArtificialSynthetic Peptide 301Asp
Ser Ser Asp Leu Ala Ser 1 5 30212PRTArtificialSynthetic Peptide
302Gln Gln Ala Trp Ser Tyr Ser Asp Ile Asp Asn Ala 1 5 10
3035PRTArtificialSynthetic Peptide 303Ser Tyr Tyr Met Ser 1 5
30416PRTArtificialSynthetic Peptide 304Ile Ile Thr Thr Ser Gly Asn
Thr Phe Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15
30510PRTArtificialSynthetic Peptide 305Thr Ser Asp Ile Phe Tyr Tyr
Arg Asn Leu 1 5 10 306366DNAArtificialSynthetic Sequence
306atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct atgatatgac ccagactcca gcctctgtgg aggtagctgt
gggaggcaca 120gtcaccatca agtgccaggc cagtgaggac atttataggt
tattggcctg gtatcaacag 180aaaccagggc agcctcccaa gctcctgatc
tatgattcat ccgatctggc atctggggtc 240ccatcgcggt tcaaaggcag
tggatctggg acagagttca ctctcgccat cagcggtgtg 300cagtgtgacg
atgctgccac ttactactgt caacaggctt ggagttatag tgatattgat 360aatgct
366307369DNAArtificialSynthetic Sequence 307atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgccgggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtagctac tacatgagct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatcattact actagtggta atacatttta
cgcgagctgg 240gcgaaaggcc ggctcaccat ctccagaacc tcgaccacgg
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagaa cttctgatat tttttattat 360cgtaacttg
36930833DNAArtificialSynthetic Sequence 308caggccagtg aggacattta
taggttattg gcc 3330921DNAArtificialSynthetic Sequence 309gattcatccg
atctggcatc t 2131036DNAArtificialSynthetic Sequence 310caacaggctt
ggagttatag tgatattgat aatgct 3631115DNAArtificialSynthetic Sequence
311agctactaca tgagc 1531248DNAArtificialSynthetic Sequence
312atcattacta ctagtggtaa tacattttac gcgagctggg cgaaaggc
4831330DNAArtificialSynthetic Sequence 313acttctgata ttttttatta
tcgtaacttg 30314123PRTArtificialSynthetic Polypeptide 314Met Asp
Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Pro Gly Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Ala Ser Pro 20
25 30 Val Ser Ala Ala Val Gly Ala Thr Val Thr Ile Asn Cys Gln Ser
Ser 35 40 45 Gln Ser Val Tyr Asn Asp Met Asp Leu Ala Trp Phe Gln
Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Ser Ala
Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr 85 90 95 Leu Thr Ile Ser Gly Val Gln
Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Leu Gly Ala Phe Asp
Asp Asp Ala Asp Asn Thr 115 120 315129PRTArtificialSynthetic
Polypeptide 315Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser Leu Thr 35 40 45 Arg His Ala Ile Thr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Cys Ile
Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Lys Gly
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Arg
Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Val Ile Gly Asp Thr Ala Gly Tyr Ala Tyr Phe Thr Gly Leu Asp
115 120 125 Leu 31613PRTArtificialSynthetic Peptide 316Gln Ser Ser
Gln Ser Val Tyr Asn Asp Met Asp Leu Ala 1 5 10
3177PRTArtificialSynthetic Peptide 317Ser Ala Ser Thr Leu Ala Ser 1
5 31811PRTArtificialSynthetic Peptide 318Leu Gly Ala Phe Asp Asp
Asp Ala Asp Asn Thr 1 5 10 3195PRTArtificialSynthetic Peptide
319Arg His Ala Ile Thr 1 5 32016PRTArtificialSynthetic Peptide
320Cys Ile Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Thr Trp Ala Lys Gly
1 5 10 15 32116PRTArtificialSynthetic Peptide 321Val Ile Gly Asp
Thr Ala Gly Tyr Ala Tyr Phe Thr Gly Leu Asp Leu 1 5 10 15
322369DNAArtificialSynthetic Sequence 322atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acgtttgcag ccgtgctgac
ccagactgca tcacccgtgt ctgccgctgt gggagccaca 120gtcaccatca
actgccagtc cagtcagagt gtttataatg acatggactt agcctggttt
180cagcagaaac cagggcagcc tcccaagctc ctgatctatt ctgcatccac
tctggcatct 240ggggtcccat cgcggttcag cggcagtgga tctgggacag
agttcactct caccatcagc 300ggcgtgcagt gtgacgatgc tgccacttac
tactgtctag gcgcttttga tgatgatgct 360gataatact
369323387DNAArtificialSynthetic Sequence 323atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cactaggcat gcaataacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg atgcatttgg agtggtggta gcacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctcag aatcaccagt 300ccgacaaccg aggacacggc cacctacttc
tgtgccagag tcattggcga tactgctggt 360tatgcttatt ttacggggct tgacttg
38732439DNAArtificialSynthetic Sequence 324cagtccagtc agagtgttta
taatgacatg gacttagcc 3932521DNAArtificialSynthetic Sequence
325tctgcatcca ctctggcatc t 2132633DNAArtificialSynthetic Sequence
326ctaggcgctt ttgatgatga tgctgataat act
3332715DNAArtificialSynthetic Sequence 327aggcatgcaa taacc
1532848DNAArtificialSynthetic Sequence 328tgcatttgga gtggtggtag
cacatactac gcgacctggg cgaaaggc 4832948DNAArtificialSynthetic
Sequence 329gtcattggcg atactgctgg ttatgcttat tttacggggc ttgacttg
48330121PRTArtificialSynthetic Polypeptide 330Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val
Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40
45 Gln Ser Val Tyr Asn Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60 Pro Pro Lys Leu Leu Ile Tyr Thr Ala Ser Ser Leu Ala Ser
Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr 85 90 95 Ile Ser Gly Val Glu Cys Ala Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr Thr Ser Asp Val Asp Asn
Val 115 120 331130PRTArtificialSynthetic Polypeptide 331Met Glu Thr
Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val
Gln Cys Gln Ser Leu Glu Glu Ala Gly Gly Arg Leu Val Thr Pro 20 25
30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser
35 40 45 Ser Tyr Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu 50 55 60 Tyr Ile Gly Ile Ile Ser Ser Ser Gly Ser Thr Tyr
Tyr Ala Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Gln Ala
Ser Ser Thr Thr Val Asp 85 90
95 Leu Lys Ile Thr Ser Pro Thr Thr Glu Asp Ser Ala Thr Tyr Phe Cys
100 105 110 Ala Arg Gly Gly Ala Gly Ser Gly Gly Val Trp Leu Leu Asp
Gly Phe 115 120 125 Asp Pro 130 33211PRTArtificialSynthetic Peptide
332Gln Ala Ser Gln Ser Val Tyr Asn Trp Leu Ser 1 5 10
3337PRTArtificialSynthetic Peptide 333Thr Ala Ser Ser Leu Ala Ser 1
5 33411PRTArtificialSynthetic Peptide 334Gln Gln Gly Tyr Thr Ser
Asp Val Asp Asn Val 1 5 10 3355PRTArtificialSynthetic Peptide
335Ser Tyr Ala Met Gly 1 5 33616PRTArtificialSynthetic Peptide
336Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Thr Trp Ala Lys Gly
1 5 10 15 33716PRTArtificialSynthetic Peptide 337Gly Gly Ala Gly
Ser Gly Gly Val Trp Leu Leu Asp Gly Phe Asp Pro 1 5 10 15
338363DNAArtificialSynthetic Sequence 338atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac
ccagactcca gcctctgtgg aggtagctgt gggaggcaca 120gtcaccatca
agtgccaggc cagtcagagt gtttataatt ggttatcctg gtatcagcag
180aaaccagggc agcctcccaa gctcctgatc tatactgcat ccagtctggc
atctggggtc 240ccatcgcggt tcagtggcag tggatctggg acagagttca
ctctcaccat cagcggcgtg 300gagtgtgccg atgctgccac ttactactgt
caacagggtt atactagtga tgttgataat 360gtt
363339390DNAArtificialSynthetic Sequence 339atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg aggccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gaatcgacct cagtagctat gcaatgggct gggtccgcca ggctccaggg
180aaggggctgg aatacatcgg aatcattagt agtagtggta gcacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctcacaagcc tcgtcgacca
cggtggatct gaaaattacc 300agtccgacaa ccgaggactc ggccacatat
ttctgtgcca gagggggtgc tggtagtggt 360ggtgtttggc tgcttgatgg
ttttgatccc 39034033DNAArtificialSynthetic Sequence 340caggccagtc
agagtgttta taattggtta tcc 3334121DNAArtificialSynthetic Sequence
341actgcatcca gtctggcatc t 2134233DNAArtificialSynthetic Sequence
342caacagggtt atactagtga tgttgataat gtt
3334315DNAArtificialSynthetic Sequence 343agctatgcaa tgggc
1534448DNAArtificialSynthetic Sequence 344atcattagta gtagtggtag
cacatactac gcgacctggg cgaaaggc 4834548DNAArtificialSynthetic
Sequence 345gggggtgctg gtagtggtgg tgtttggctg cttgatggtt ttgatccc
48346123PRTArtificialSynthetic Polypeptide 346Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Lys Cys Ala Asp Val Val Met Thr Gln Thr Pro Ala 20 25 30 Ser
Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala 35 40
45 Ser Glu Asn Ile Tyr Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly
50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Thr Val Gly Asp Leu Ala
Ser Gly 65 70 75 80 Val Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr
Glu Phe Thr Leu 85 90 95 Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala
Ala Thr Tyr Tyr Cys Gln 100 105 110 Gln Gly Tyr Ser Ser Ser Tyr Val
Asp Asn Val 115 120 347130PRTArtificialSynthetic Polypeptide 347Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Arg Leu Val Thr
20 25 30 Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe
Ser Leu 35 40 45 Asn Asp Tyr Ala Val Gly Trp Phe Arg Gln Ala Pro
Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Arg Ser Ser Gly
Thr Thr Ala Tyr Ala Thr 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile
Ser Ala Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Ile Thr Ser Pro
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Gly Gly
Ala Gly Ser Ser Gly Val Trp Ile Leu Asp Gly Phe 115 120 125 Ala Pro
130 34811PRTArtificialSynthetic Peptide 348Gln Ala Ser Glu Asn Ile
Tyr Asn Trp Leu Ala 1 5 10 3497PRTArtificialSynthetic Peptide
349Thr Val Gly Asp Leu Ala Ser 1 5 35012PRTArtificialSynthetic
Peptide 350Gln Gln Gly Tyr Ser Ser Ser Tyr Val Asp Asn Val 1 5 10
3515PRTArtificialSynthetic Peptide 351Asp Tyr Ala Val Gly 1 5
35216PRTArtificialSynthetic Peptide 352Tyr Ile Arg Ser Ser Gly Thr
Thr Ala Tyr Ala Thr Trp Ala Lys Gly 1 5 10 15
35316PRTArtificialSynthetic Peptide 353Gly Gly Ala Gly Ser Ser Gly
Val Trp Ile Leu Asp Gly Phe Ala Pro 1 5 10 15
354369DNAArtificialSynthetic Sequence 354atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60aaatgtgccg atgttgtgat
gacccagact ccagcctccg tgtctgcagc tgtgggaggc 120acagtcacca
tcaattgcca ggccagtgag aacatttata attggttagc ctggtatcag
180cagaaaccag ggcagcctcc caagctcctg atctatactg taggcgatct
ggcatctggg 240gtctcatcgc ggttcaaagg cagtggatct gggacagagt
tcactctcac catcagcgac 300ctggagtgtg ccgatgctgc cacttactat
tgtcaacagg gttatagtag tagttatgtt 360gataatgtt
369355390DNAArtificialSynthetic Sequence 355atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60gagcagctga aggagtccgg
gggtcgcctg gtcacgcctg ggacacccct gacactcacc 120tgcacagtct
ctggattctc cctcaatgac tatgcagtgg gctggttccg ccaggctcca
180gggaaggggc tggaatggat cggatacatt cgtagtagtg gtaccacagc
ctacgcgacc 240tgggcgaaag gccgattcac catctccgct acctcgacca
cggtggatct gaaaatcacc 300agtccgacaa ccgaggacac ggccacctat
ttctgtgcca gagggggtgc tggtagtagt 360ggtgtgtgga tccttgatgg
ttttgctccc 39035633DNAArtificialSynthetic Sequence 356caggccagtg
agaacattta taattggtta gcc 3335721DNAArtificialSynthetic Sequence
357actgtaggcg atctggcatc t 2135836DNAArtificialSynthetic Sequence
358caacagggtt atagtagtag ttatgttgat aatgtt
3635915DNAArtificialSynthetic Sequence 359gactatgcag tgggc
1536048DNAArtificialSynthetic Sequence 360tacattcgta gtagtggtac
cacagcctac gcgacctggg cgaaaggc 4836148DNAArtificialSynthetic
Sequence 361gggggtgctg gtagtagtgg tgtgtggatc cttgatggtt ttgctccc
48362121PRTArtificialSynthetic Polypeptide 362Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Ser 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40
45 Gln Ser Val Tyr Gln Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro
50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ala Thr Leu
Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly
Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Leu Glu Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Ala Tyr Arg Asp Val Asp
Ser 115 120 363130PRTArtificialSynthetic Polypeptide 363Met Glu Thr
Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val
Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25
30 Gly Ala Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Thr
35 40 45 Ser Thr Tyr Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60 Glu Trp Ile Ala Cys Ile Asp Ala Gly Ser Ser Gly
Ser Thr Tyr Tyr 65 70 75 80 Ala Thr Trp Val Asn Gly Arg Phe Thr Ile
Ser Lys Thr Ser Ser Thr 85 90 95 Thr Val Thr Leu Gln Met Thr Ser
Leu Thr Ala Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Lys Trp
Asp Tyr Gly Gly Asn Val Gly Trp Gly Tyr 115 120 125 Asp Leu 130
36413PRTArtificialSynthetic Peptide 364Gln Ala Ser Gln Ser Val Tyr
Gln Asn Asn Tyr Leu Ser 1 5 10 3657PRTArtificialSynthetic Peptide
365Gly Ala Ala Thr Leu Ala Ser 1 5 3669PRTArtificialSynthetic
Peptide 366Ala Gly Ala Tyr Arg Asp Val Asp Ser 1 5
3676PRTArtificialSynthetic Peptide 367Ser Thr Tyr Tyr Ile Tyr 1 5
36818PRTArtificialSynthetic Peptide 368Cys Ile Asp Ala Gly Ser Ser
Gly Ser Thr Tyr Tyr Ala Thr Trp Val 1 5 10 15 Asn Gly
36913PRTArtificialSynthetic Peptide 369Trp Asp Tyr Gly Gly Asn Val
Gly Trp Gly Tyr Asp Leu 1 5 10 370363DNAArtificialSynthetic
Sequence 370atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgctc aagtgctgac ccagactcca tcctccgtgt ctgcagctgt
gggaggcaca 120gtcaccatca attgccaggc cagtcagagt gtttatcaga
acaactactt atcctggttt 180cagcagaaac cagggcagcc tcccaagctc
ctgatctatg gtgcggccac tctggcatct 240ggggtcccat cgcggttcaa
aggcagtgga tctgggacac agttcactct caccatcagc 300gacctggagt
gtgacgatgc tgccacttac tactgtgcag gcgcttatag ggatgtggat 360tct
363371390DNAArtificialSynthetic Sequence 371atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgttggagg agtccggggg
agacctggtc aagcctgggg catccctgac actcacctgc 120acagcctctg
gattctcctt tactagtacc tactacatct actgggtccg ccaggctcca
180gggaaggggc tggagtggat cgcatgtatt gatgctggta gtagtggtag
cacttactac 240gcgacctggg tgaatggccg attcaccatc tccaaaacct
cgtcgaccac ggtgactctg 300caaatgacca gtctgacagc cgcggacacg
gccacctatt tctgtgcgaa atgggattat 360ggtggtaatg ttggttgggg
ttatgacttg 39037239DNAArtificialSynthetic Sequence 372caggccagtc
agagtgttta tcagaacaac tacttatcc 3937321DNAArtificialSynthetic
Sequence 373ggtgcggcca ctctggcatc t 2137427DNAArtificialSynthetic
Sequence 374gcaggcgctt atagggatgt ggattct
2737518DNAArtificialSynthetic Sequence 375agtacctact acatctac
1837654DNAArtificialSynthetic Sequence 376tgtattgatg ctggtagtag
tggtagcact tactacgcga cctgggtgaa tggc 5437739DNAArtificialSynthetic
Sequence 377tgggattatg gtggtaatgt tggttggggt tatgacttg
39378120PRTArtificialSynthetic Polypeptide 378Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys Ala Phe Glu Leu Thr Gln Thr Pro Ser Ser 20 25 30 Val
Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40
45 Gln Ser Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60 Pro Pro Lys Phe Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser
Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala
Thr Tyr Tyr Cys Gln Ser 100 105 110 Tyr Tyr Asp Ser Val Ser Asn Pro
115 120 379127PRTArtificialSynthetic Polypeptide 379Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25 30
Glu Gly Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Leu Asp Leu Gly 35
40 45 Thr Tyr Trp Phe Met Cys Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 50 55 60 Glu Trp Ile Ala Cys Ile Tyr Thr Gly Ser Ser Gly Ser
Thr Phe Tyr 65 70 75 80 Ala Ser Trp Val Asn Gly Arg Phe Thr Ile Ser
Lys Thr Ser Ser Thr 85 90 95 Thr Val Thr Leu Gln Met Thr Ser Leu
Thr Ala Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Gly Tyr
Ser Gly Tyr Gly Tyr Phe Lys Leu 115 120 125
38011PRTArtificialSynthetic Peptide 380Gln Ala Ser Gln Ser Ile Ser
Ser Tyr Leu Ala 1 5 10 3817PRTArtificialSynthetic Peptide 381Arg
Ala Ser Thr Leu Ala Ser 1 5 38210PRTArtificialSynthetic Peptide
382Gln Ser Tyr Tyr Asp Ser Val Ser Asn Pro 1 5 10
3836PRTArtificialSynthetic Peptide 383Thr Tyr Trp Phe Met Cys 1 5
38418PRTArtificialSynthetic Peptide 384Cys Ile Tyr Thr Gly Ser Ser
Gly Ser Thr Phe Tyr Ala Ser Trp Val 1 5 10 15 Asn Gly
38510PRTArtificialSynthetic Peptide 385Gly Tyr Ser Gly Tyr Gly Tyr
Phe Lys Leu 1 5 10 386360DNAArtificialSynthetic Sequence
386atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcat tcgaattgac ccagactcca tcctccgtgg aggcagctgt
gggaggcaca 120gtcaccatca agtgccaggc cagtcagagc attagtagtt
acttagcctg gtatcagcag 180aaaccagggc agcctcccaa gttcctgatc
tacagggcgt ccactctggc atctggggtc 240ccatcgcgat tcaaaggcag
tggatctggg acagagttca ctctcaccat cagcgacctg 300gagtgtgccg
atgctgccac ttactactgt caaagctatt atgatagtgt ttcaaatcct
360387381DNAArtificialSynthetic Sequence 387atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgttggagg agtccggggg
agacctggtc aagcctgagg gatccctgac actcacctgc 120aaagcctctg
gactcgacct cggtacctac tggttcatgt gctgggtccg ccaggctcca
180gggaaggggc tggagtggat cgcttgtatt tatactggta gtagtggttc
cactttctac 240gcgagctggg tgaatggccg attcaccatc tccaaaacct
cgtcgaccac ggtgactctg 300caaatgacca gtctgacagc cgcggacacg
gccacttatt tttgtgcgag aggttatagt 360ggttatggtt attttaagtt g
38138833DNAArtificialSynthetic Sequence 388caggccagtc agagcattag
tagttactta gcc 3338921DNAArtificialSynthetic Sequence 389agggcgtcca
ctctggcatc t 2139030DNAArtificialSynthetic Sequence 390caaagctatt
atgatagtgt ttcaaatcct 3039118DNAArtificialSynthetic Sequence
391acctactggt tcatgtgc 1839254DNAArtificialSynthetic Sequence
392tgtatttata ctggtagtag tggttccact ttctacgcga gctgggtgaa tggc
5439330DNAArtificialSynthetic Sequence 393ggttatagtg gttatggtta
ttttaagttg 30394124PRTArtificialSynthetic Polypeptide 394Met Asp
Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Pro Gly Val Thr Phe Ala Ile Glu Met Thr Gln Ser Pro Phe Ser 20
25 30 Val Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala
Ser 35 40 45 Gln Ser Val Tyr Lys Asn Asn Gln Leu Ser Trp Tyr Gln
Gln Lys Ser 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala
Ser Ala Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser
Gly Ser Gly Thr Glu Phe Thr 85 90 95 Leu Thr Ile Ser Asp Val Gln
Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Ala Ile Thr
Gly Ser Ile Asp Thr Asp Gly 115 120
395130PRTArtificialSynthetic Polypeptide 395Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Asp Leu Val Lys Pro 20 25 30 Gly Ala
Ser Leu Thr Leu Thr Cys Thr Thr Ser Gly Phe Ser Phe Ser 35 40 45
Ser Ser Tyr Phe Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50
55 60 Glu Trp Ile Ala Cys Ile Tyr Gly Gly Asp Gly Ser Thr Tyr Tyr
Ala 65 70 75 80 Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
Ser Thr Thr 85 90 95 Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala
Asp Thr Ala Thr Tyr 100 105 110 Phe Cys Ala Arg Glu Trp Ala Tyr Ser
Gln Gly Tyr Phe Gly Ala Phe 115 120 125 Asp Leu 130
39613PRTArtificialSynthetic Peptide 396Gln Ala Ser Gln Ser Val Tyr
Lys Asn Asn Gln Leu Ser 1 5 10 3977PRTArtificialSynthetic Peptide
397Gly Ala Ser Ala Leu Ala Ser 1 5 39812PRTArtificialSynthetic
Peptide 398Ala Gly Ala Ile Thr Gly Ser Ile Asp Thr Asp Gly 1 5 10
3996PRTArtificialSynthetic Peptide 399Ser Ser Tyr Phe Ile Cys 1 5
40017PRTArtificialSynthetic Peptide 400Cys Ile Tyr Gly Gly Asp Gly
Ser Thr Tyr Tyr Ala Ser Trp Ala Lys 1 5 10 15 Gly
40114PRTArtificialSynthetic Peptide 401Glu Trp Ala Tyr Ser Gln Gly
Tyr Phe Gly Ala Phe Asp Leu 1 5 10 402372DNAArtificialSynthetic
Sequence 402atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgtc 60acatttgcca tcgaaatgac ccagagtcca ttctccgtgt ctgcagctgt
gggaggcaca 120gtcagcatca gttgccaggc cagtcagagt gtttataaga
acaaccaatt atcctggtat 180cagcagaaat cagggcagcc tcccaagctc
ctgatctatg gtgcatcggc tctggcatct 240ggggtcccat cgcggttcaa
aggcagtgga tctgggacag agttcactct caccatcagc 300gacgtgcagt
gtgacgatgc tgccacttac tactgtgcag gcgctattac tggtagtatt
360gatacggatg gt 372403390DNAArtificialSynthetic Sequence
403atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgttggagg agtccggggg agacctggtc aagcctgggg catccctgac
actcacctgc 120acaacttctg gattctcctt cagtagcagc tacttcattt
gctgggtccg ccaggctcca 180gggaaggggc tggagtggat cgcatgcatt
tatggtggtg atggcagcac atactacgcg 240agctgggcga aaggccgatt
caccatctcc aaaacctcgt cgaccacggt gacgctgcaa 300atgaccagtc
tgacagccgc ggacacggcc acctatttct gtgcgagaga atgggcatat
360agtcaaggtt attttggtgc ttttgatctc 39040439DNAArtificialSynthetic
Sequence 404caggccagtc agagtgttta taagaacaac caattatcc
3940521DNAArtificialSynthetic Sequence 405ggtgcatcgg ctctggcatc t
2140636DNAArtificialSynthetic Sequence 406gcaggcgcta ttactggtag
tattgatacg gatggt 3640718DNAArtificialSynthetic Sequence
407agcagctact tcatttgc 1840851DNAArtificialSynthetic Sequence
408tgcatttatg gtggtgatgg cagcacatac tacgcgagct gggcgaaagg c
5140942DNAArtificialSynthetic Sequence 409gaatgggcat atagtcaagg
ttattttggt gcttttgatc tc 42410124PRTArtificialSynthetic Polypeptide
410Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Pro Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro
Ala Ser 20 25 30 Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys
Cys Gln Ala Ser 35 40 45 Glu Asp Ile Ser Ser Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile Tyr Ala
Ala Ser Asn Leu Glu Ser Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly
Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr 85 90 95 Ile Ser Asp Leu
Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys 100 105 110 Thr Tyr
Gly Thr Ile Ser Ile Ser Asp Gly Asn Ala 115 120
411124PRTArtificialSynthetic Polypeptide 411Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40 45
Ser Tyr Phe Met Thr Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu 50
55 60 Tyr Ile Gly Phe Ile Asn Pro Gly Gly Ser Ala Tyr Tyr Ala Ser
Trp 65 70 75 80 Val Lys Gly Arg Phe Thr Ile Ser Lys Ser Ser Thr Thr
Val Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Val Leu Ile Val Ser Tyr Gly Ala
Phe Thr Ile 115 120 41211PRTArtificialSynthetic Peptide 412Gln Ala
Ser Glu Asp Ile Ser Ser Tyr Leu Ala 1 5 10
4137PRTArtificialSynthetic Peptide 413Ala Ala Ser Asn Leu Glu Ser 1
5 41414PRTArtificialSynthetic Peptide 414Gln Cys Thr Tyr Gly Thr
Ile Ser Ile Ser Asp Gly Asn Ala 1 5 10 4155PRTArtificialSynthetic
Peptide 415Ser Tyr Phe Met Thr 1 5 41616PRTArtificialSynthetic
Peptide 416Phe Ile Asn Pro Gly Gly Ser Ala Tyr Tyr Ala Ser Trp Val
Lys Gly 1 5 10 15 41711PRTArtificialSynthetic Peptide 417Val Leu
Ile Val Ser Tyr Gly Ala Phe Thr Ile 1 5 10
418372DNAArtificialSynthetic Sequence 418atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgatg ttgtgatgac
ccagactcca gcctccgtgg aggcagctgt gggaggcaca 120gtcaccatca
agtgccaggc cagtgaggat attagtagct acttagcctg gtatcagcag
180aaaccagggc agcctcccaa gctcctgatc tatgctgcat ccaatctgga
atctggggtc 240tcatcgcgat tcaaaggcag tggatctggg acagagtaca
ctctcaccat cagcgacctg 300gagtgtgccg atgctgccac ctattactgt
caatgtactt atggtactat ttctattagt 360gatggtaatg ct
372419372DNAArtificialSynthetic Sequence 419atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccaatgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cagtagctac ttcatgacct gggtccgcca ggctccaggg
180gaggggctgg aatacatcgg attcattaat cctggtggta gcgcttacta
cgcgagctgg 240gtgaaaggcc gattcaccat ctccaagtcc tcgaccacgg
tagatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccaggg ttctgattgt ttcttatgga 360gcctttacca tc
37242033DNAArtificialSynthetic Sequence 420caggccagtg aggatattag
tagctactta gcc 3342121DNAArtificialSynthetic Sequence 421gctgcatcca
atctggaatc t 2142242DNAArtificialSynthetic Sequence 422caatgtactt
atggtactat ttctattagt gatggtaatg ct 4242315DNAArtificialSynthetic
Sequence 423agctacttca tgacc 1542448DNAArtificialSynthetic Sequence
424ttcattaatc ctggtggtag cgcttactac gcgagctggg tgaaaggc
4842533DNAArtificialSynthetic Sequence 425gttctgattg tttcttatgg
agcctttacc atc 33426124PRTArtificialSynthetic Polypeptide 426Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser
20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln
Ala Ser 35 40 45 Glu Asp Ile Glu Ser Tyr Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser
Asn Leu Glu Ser Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly
Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys 100 105 110 Thr Tyr Gly Ile
Ile Ser Ile Ser Asp Gly Asn Ala 115 120
427124PRTArtificialSynthetic Polypeptide 427Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40 45
Ser Tyr Phe Met Thr Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu 50
55 60 Tyr Ile Gly Phe Met Asn Thr Gly Asp Asn Ala Tyr Tyr Ala Ser
Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Val Leu Val Val Ala Tyr Gly Ala
Phe Asn Ile 115 120 42811PRTArtificialSynthetic Peptide 428Gln Ala
Ser Glu Asp Ile Glu Ser Tyr Leu Ala 1 5 10
4297PRTArtificialSynthetic Peptide 429Gly Ala Ser Asn Leu Glu Ser 1
5 43014PRTArtificialSynthetic Peptide 430Gln Cys Thr Tyr Gly Ile
Ile Ser Ile Ser Asp Gly Asn Ala 1 5 10 4315PRTArtificialSynthetic
Peptide 431Ser Tyr Phe Met Thr 1 5 43216PRTArtificialSynthetic
Peptide 432Phe Met Asn Thr Gly Asp Asn Ala Tyr Tyr Ala Ser Trp Ala
Lys Gly 1 5 10 15 43311PRTArtificialSynthetic Peptide 433Val Leu
Val Val Ala Tyr Gly Ala Phe Asn Ile 1 5 10
434372DNAArtificialSynthetic Sequence 434atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgatg ttgtgatgac
ccagactcca gcctccgtgt ctgcagctgt gggaggcaca 120gtcaccatca
agtgccaggc cagtgaggac attgaaagct atctagcctg gtatcagcag
180aaaccagggc agcctcccaa gctcctgatc tatggtgcat ccaatctgga
atctggggtc 240tcatcgcggt tcaaaggcag tggatctggg acagagttca
ctctcaccat cagcgacctg 300gagtgtgccg atgctgccac ttactattgt
caatgcactt atggtattat tagtattagt 360gatggtaatg ct
372435372DNAArtificialSynthetic Sequence 435atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtgtctg
gattctccct cagtagctac ttcatgacct gggtccgcca ggctccaggg
180gaggggctgg aatacatcgg attcatgaat actggtgata acgcatacta
cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccaggg ttcttgttgt tgcttatgga 360gcctttaaca tc
37243633DNAArtificialSynthetic Sequence 436caggccagtg aggacattga
aagctatcta gcc 3343721DNAArtificialSynthetic Sequence 437ggtgcatcca
atctggaatc t 2143842DNAArtificialSynthetic Sequence 438caatgcactt
atggtattat tagtattagt gatggtaatg ct 4243915DNAArtificialSynthetic
Sequence 439agctacttca tgacc 1544048DNAArtificialSynthetic Sequence
440ttcatgaata ctggtgataa cgcatactac gcgagctggg cgaaaggc
4844133DNAArtificialSynthetic Sequence 441gttcttgttg ttgcttatgg
agcctttaac atc 33442124PRTArtificialSynthetic Polypeptide 442Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro
20 25 30 Val Ser Glu Pro Val Gly Gly Thr Val Ser Ile Ser Cys Gln
Ser Ser 35 40 45 Lys Ser Val Met Asn Asn Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly
Ala Ser Asn Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Val
Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln Gly Gly Tyr
Thr Gly Tyr Ser Asp His Gly Thr 115 120
443127PRTArtificialSynthetic Polypeptide 443Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Val Glu Glu Ser Gly Gly Arg Leu Val Lys Pro 20 25 30 Asp Glu
Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35 40 45
Ser Tyr Pro Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50
55 60 Trp Ile Gly Phe Ile Asn Thr Gly Gly Thr Ile Val Tyr Ala Ser
Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Gly Ser Tyr Val Ser Ser Gly Tyr
Ala Tyr Tyr Phe Asn Val 115 120 125 44413PRTArtificialSynthetic
Peptide 444Gln Ser Ser Lys Ser Val Met Asn Asn Asn Tyr Leu Ala 1 5
10 4457PRTArtificialSynthetic Peptide 445Gly Ala Ser Asn Leu Ala
Ser 1 5 44612PRTArtificialSynthetic Peptide 446Gln Gly Gly Tyr Thr
Gly Tyr Ser Asp His Gly Thr 1 5 10 4475PRTArtificialSynthetic
Peptide 447Ser Tyr Pro Met Asn 1 5 44816PRTArtificialSynthetic
Peptide 448Phe Ile Asn Thr Gly Gly Thr Ile Val Tyr Ala Ser Trp Ala
Lys Gly 1 5 10 15 44914PRTArtificialSynthetic Peptide 449Gly Ser
Tyr Val Ser Ser Gly Tyr Ala Tyr Tyr Phe Asn Val 1 5 10
450372DNAArtificialSynthetic Sequence 450atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgccg ccgtgctgac
ccagactcca tctcccgtgt ctgaacctgt gggaggcaca 120gtcagcatca
gttgccagtc cagtaagagt gttatgaata acaactactt agcctggtat
180cagcagaaac cagggcagcc tcccaagctc ctgatctatg gtgcatccaa
tctggcatct 240ggggtcccat cacggttcag cggcagtgga tctgggacac
agttcactct caccatcagc 300gacgtgcagt gtgacgatgc tgccacttac
tactgtcaag gcggttatac tggttatagt 360gatcatggga ct
372451381DNAArtificialSynthetic Sequence 451atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc aagcctgacg aaaccctgac actcacctgc 120acagtctctg
gaatcgacct cagtagctat ccaatgaact gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg attcattaat actggtggta ccatagtcta
cgcgagctgg 240gcaaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag gcagttatgt ttcatctggt 360tatgcctact attttaatgt c
38145239DNAArtificialSynthetic Sequence 452cagtccagta agagtgttat
gaataacaac tacttagcc 3945321DNAArtificialSynthetic Sequence
453ggtgcatcca atctggcatc t 2145436DNAArtificialSynthetic Sequence
454caaggcggtt atactggtta tagtgatcat gggact
3645515DNAArtificialSynthetic Sequence 455agctatccaa tgaac
1545648DNAArtificialSynthetic Sequence 456ttcattaata ctggtggtac
catagtctac gcgagctggg caaaaggc 4845742DNAArtificialSynthetic
Sequence 457ggcagttatg tttcatctgg ttatgcctac tattttaatg tc
42458121PRTArtificialSynthetic Polypeptide 458Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5
10 15 Leu Pro Gly Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser
Pro 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys
Gln Ser Ser 35 40 45 Gln Ser Val Tyr Asn Asn Asn Trp Leu Ser Trp
Phe Gln Gln Lys Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr
Lys Ala Ser Thr Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys
Gly Ser Gly Ser Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp
Val Gln Cys Asp Asp Val Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Gly
Tyr Leu Asp Ser Val Ile 115 120 459126PRTArtificialSynthetic
Polypeptide 459Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser Leu Ser 35 40 45 Thr Tyr Ser Ile Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile
Ala Asn Ser Gly Thr Thr Phe Tyr Ala Asn Trp 65 70 75 80 Ala Lys Gly
Arg Phe Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys
Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105
110 Arg Glu Ser Gly Met Tyr Asn Glu Tyr Gly Lys Phe Asn Ile 115 120
125 46013PRTArtificialSynthetic Peptide 460Gln Ser Ser Gln Ser Val
Tyr Asn Asn Asn Trp Leu Ser 1 5 10 4617PRTArtificialSynthetic
Peptide 461Lys Ala Ser Thr Leu Ala Ser 1 5
4629PRTArtificialSynthetic Peptide 462Ala Gly Gly Tyr Leu Asp Ser
Val Ile 1 5 4635PRTArtificialSynthetic Peptide 463Thr Tyr Ser Ile
Asn 1 5 46416PRTArtificialSynthetic Peptide 464Ile Ile Ala Asn Ser
Gly Thr Thr Phe Tyr Ala Asn Trp Ala Lys Gly 1 5 10 15
46513PRTArtificialSynthetic Peptide 465Glu Ser Gly Met Tyr Asn Glu
Tyr Gly Lys Phe Asn Ile 1 5 10 466363DNAArtificialSynthetic
Sequence 466atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgccg ccgtgctgac ccagactcca tctcccgtgt ctgcagctgt
gggaggcaca 120gtcagcatca gttgccagtc cagtcagagt gtttataata
acaactggtt atcctggttt 180cagcagaaac cagggcagcc tcccaagctc
ctgatctaca aggcatccac tctggcatct 240ggggtcccat cgcggttcaa
aggcagtgga tctgggacac agttcactct caccatcagc 300gacgtgcagt
gtgacgatgt tgccacttac tactgtgcgg gcggttatct tgatagtgtt 360att
363467378DNAArtificialSynthetic Sequence 467atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cagtacctat tcaataaact gggtccgcca ggctccaggg
180aagggcctgg aatggatcgg aatcattgct aatagtggta ccacattcta
cgcgaactgg 240gcgaaaggcc gattcaccgt ctccaaaacc tcgaccacgg
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag agagtggaat gtacaatgaa 360tatggtaaat ttaacatc
37846839DNAArtificialSynthetic Sequence 468cagtccagtc agagtgttta
taataacaac tggttatcc 3946921DNAArtificialSynthetic Sequence
469aaggcatcca ctctggcatc t 2147027DNAArtificialSynthetic Sequence
470gcgggcggtt atcttgatag tgttatt 2747115DNAArtificialSynthetic
Sequence 471acctattcaa taaac 1547248DNAArtificialSynthetic Sequence
472atcattgcta atagtggtac cacattctac gcgaactggg cgaaaggc
4847339DNAArtificialSynthetic Sequence 473gagagtggaa tgtacaatga
atatggtaaa tttaacatc 39474122PRTArtificialSynthetic Polypeptide
474Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Ser Asp Met Thr Gln Thr Pro
Ser Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn
Cys Gln Ala Ser 35 40 45 Glu Asn Ile Tyr Ser Phe Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile Phe Lys
Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu
Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Ala
Thr Val Tyr Asp Ile Asp Asn Asn 115 120
475128PRTArtificialSynthetic Polypeptide 475Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35 40 45
Ala Tyr Ala Met Ile Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu 50
55 60 Trp Ile Thr Ile Ile Tyr Pro Asn Gly Ile Thr Tyr Tyr Ala Asn
Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Val Ser Lys Thr Ser Thr Ala
Met Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Ala Glu Ser Ser Lys Asn Ala
Tyr Trp Gly Tyr Phe Asn Val 115 120 125 47611PRTArtificialSynthetic
Peptide 476Gln Ala Ser Glu Asn Ile Tyr Ser Phe Leu Ala 1 5 10
4777PRTArtificialSynthetic Peptide 477Lys Ala Ser Thr Leu Ala Ser 1
5 47812PRTArtificialSynthetic Peptide 478Gln Gln Gly Ala Thr Val
Tyr Asp Ile Asp Asn Asn 1 5 10 4795PRTArtificialSynthetic Peptide
479Ala Tyr Ala Met Ile 1 5 48016PRTArtificialSynthetic Peptide
480Ile Ile Tyr Pro Asn Gly Ile Thr Tyr Tyr Ala Asn Trp Ala Lys Gly
1 5 10 15 48115PRTArtificialSynthetic Peptide 481Asp Ala Glu Ser
Ser Lys Asn Ala Tyr Trp Gly Tyr Phe Asn Val 1 5 10 15
482366DNAArtificialSynthetic Sequence 482atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct ctgatatgac
ccagactcca tcctccgtgt ctgcagctgt gggaggcaca 120gtcaccatca
attgccaggc cagtgagaac atttatagct ttttggcctg gtatcagcag
180aaaccagggc agcctcccaa gctcctgatc ttcaaggctt ccactctggc
atctggggtc 240tcatcgcggt tcaaaggcag tggatctggg acacagttca
ctctcaccat cagcgacctg 300gagtgtgacg atgctgccac ttactactgt
caacagggtg ctactgtgta tgatattgat 360aataat
366483384DNAArtificialSynthetic Sequence 483atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtttctg
gaatcgacct cagtgcctat gcaatgatct gggtccgcca ggctccaggg
180gaggggctgg aatggatcac aatcatttat cctaatggta tcacatacta
cgcgaactgg 240gcgaaaggcc gattcaccgt ctccaaaacc tcgaccgcga
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag atgcagaaag tagtaagaat 360gcttattggg gctactttaa cgtc
38448433DNAArtificialSynthetic Sequence 484caggccagtg agaacattta
tagctttttg gcc 3348521DNAArtificialSynthetic Sequence 485aaggcttcca
ctctggcatc t 2148636DNAArtificialSynthetic Sequence 486caacagggtg
ctactgtgta tgatattgat aataat 3648715DNAArtificialSynthetic Sequence
487gcctatgcaa tgatc 1548848DNAArtificialSynthetic Sequence
488atcatttatc ctaatggtat cacatactac gcgaactggg cgaaaggc
4848945DNAArtificialSynthetic Sequence 489gatgcagaaa gtagtaagaa
tgcttattgg ggctacttta acgtc 45490122PRTArtificialSynthetic
Polypeptide 490Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Ser Asp Met Thr
Gln Thr Pro Ser Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val
Thr Ile Asn Cys Gln Ala Ser 35 40 45 Glu Asn Ile Tyr Ser Phe Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu
Ile Phe Arg Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Ser Ser Arg
Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr 85 90 95 Ile
Ser Asp Leu Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105
110 Gly Ala Thr Val Tyr Asp Ile Asp Asn Asn 115 120
491128PRTArtificialSynthetic Polypeptide 491Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser 35 40 45
Ala Tyr Ala Met Ile Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu 50
55 60 Trp Ile Thr Ile Ile Tyr Pro Asn Gly Ile Thr Tyr Tyr Ala Asn
Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Val Ser Lys Thr Ser Thr Ala
Met Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Ala Glu Ser Ser Lys Asn Ala
Tyr Trp Gly Tyr Phe Asn Val 115 120 125 49211PRTArtificialSynthetic
Peptide 492Gln Ala Ser Glu Asn Ile Tyr Ser Phe Leu Ala 1 5 10
4937PRTArtificialSynthetic Peptide 493Arg Ala Ser Thr Leu Ala Ser 1
5 49412PRTArtificialSynthetic Peptide 494Gln Gln Gly Ala Thr Val
Tyr Asp Ile Asp Asn Asn 1 5 10 4955PRTArtificialSynthetic Peptide
495Ala Tyr Ala Met Ile 1 5 49616PRTArtificialSynthetic Peptide
496Ile Ile Tyr Pro Asn Gly Ile Thr Tyr Tyr Ala Asn Trp Ala Lys Gly
1 5 10 15 49715PRTArtificialSynthetic Peptide 497Asp Ala Glu Ser
Ser Lys Asn Ala Tyr Trp Gly Tyr Phe Asn Val 1 5 10 15
498366DNAArtificialSynthetic Sequence 498atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct ctgatatgac
ccagactcca tcctccgtgt ctgcagctgt gggaggcaca 120gtcaccatca
attgccaggc cagtgagaac atttatagct ttttggcctg gtatcagcag
180aaaccagggc agcctcccaa gctcctgatc ttcagggctt ccactctggc
atctggggtc 240tcatcgcggt tcaaaggcag tggatctggg acacagttca
ctctcaccat cagcgacctg 300gagtgtgacg atgctgccac ttactactgt
caacagggtg ctactgtgta tgatattgat 360aataat
366499384DNAArtificialSynthetic Sequence 499atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtttctg
gaatcgacct cagtgcctat gcaatgatct gggtccgcca ggctccaggg
180gaggggctgg aatggatcac aatcatttat cctaatggta tcacatacta
cgcgaactgg 240gcgaaaggcc gattcaccgt ctccaaaacc tcgaccgcga
tggatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag atgcagaaag tagtaagaat 360gcttattggg gctactttaa cgtc
38450033DNAArtificialSynthetic Sequence 500caggccagtg agaacattta
tagctttttg gcc 3350121DNAArtificialSynthetic Sequence 501agggcttcca
ctctggcatc t 2150236DNAArtificialSynthetic Sequence 502caacagggtg
ctactgtgta tgatattgat aataat 3650315DNAArtificialSynthetic Sequence
503gcctatgcaa tgatc 1550448DNAArtificialSynthetic Sequence
504atcatttatc ctaatggtat cacatactac gcgaactggg cgaaaggc
4850545DNAArtificialSynthetic Sequence 505gatgcagaaa gtagtaagaa
tgcttattgg ggctacttta acgtc 45506124PRTArtificialSynthetic
Polypeptide 506Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Thr Phe Ala Ile Glu Met Thr
Gln Thr Pro Ser Pro 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val
Thr Ile Asn Cys Gln Ala Ser 35 40 45 Glu Ser Val Phe Asn Asn Met
Leu Ser Trp Tyr Gln Gln Lys Pro Gly 50 55 60 His Ser Pro Lys Leu
Leu Ile Tyr Asp Ala Ser Asp Leu Ala Ser Gly 65 70 75 80 Val Pro Ser
Arg Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu 85 90 95 Thr
Ile Ser Gly Val Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala 100 105
110 Gly Tyr Lys Ser Asp Ser Asn Asp Gly Asp Asn Val 115 120
507123PRTArtificialSynthetic Polypeptide 507Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Asn 35 40 45
Arg Asn Ser Ile Thr Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu 50
55 60 Trp Ile Gly Ile Ile Thr Gly Ser Gly Arg Thr Tyr Tyr Ala Asn
Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Gly His Pro Gly Leu Gly Ser Gly
Asn Ile 115 120 50812PRTArtificialSynthetic Peptide 508Gln Ala Ser
Glu Ser Val Phe Asn Asn Met Leu Ser 1 5 10
5097PRTArtificialSynthetic Peptide 509Asp Ala Ser Asp Leu Ala Ser 1
5 51013PRTArtificialSynthetic Peptide 510Ala Gly Tyr Lys Ser Asp
Ser Asn Asp Gly Asp Asn Val 1 5 10 5115PRTArtificialSynthetic
Peptide 511Arg Asn Ser Ile Thr 1 5 51216PRTArtificialSynthetic
Peptide 512Ile Ile Thr Gly Ser Gly Arg Thr Tyr Tyr Ala Asn Trp Ala
Lys Gly 1 5 10 15 51310PRTArtificialSynthetic Peptide 513Gly His
Pro Gly Leu Gly Ser Gly Asn Ile 1 5 10 514372DNAArtificialSynthetic
Sequence 514atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgcca ttgaaatgac ccagactcca tcccccgtgt ctgccgctgt
gggaggcaca 120gtcaccatca attgccaggc cagtgagagt gtttttaata
atatgttatc ctggtatcag 180cagaaaccag ggcactctcc taagctcctg
atctatgatg catccgatct ggcatctggg 240gtcccatcgc ggttcaaagg
cagtggatct gggacacagt tcactctcac catcagtggc 300gtggagtgtg
acgatgctgc cacttactat tgtgcagggt ataaaagtga tagtaatgat
360ggcgataatg tt 372515369DNAArtificialSynthetic Sequence
515atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt
ccagtgtcag 60tcgctggagg agtccggggg tcgcctggtc acgcctggga cacccctgac
actcacctgc 120acagtctctg gattctccct caacaggaat tcaataacct
gggtccgcca ggctccaggg 180gaggggctgg aatggatcgg aatcattact
ggtagtggta gaacgtacta cgcgaactgg 240gcaaaaggcc gattcaccat
ctccaaaacc tcgaccacgg tggatctgaa aatgaccagt 300ccgacaaccg
aggacacggc cacctatttc tgtgccagag gccatcctgg tcttggtagt 360ggtaacatc
36951636DNAArtificialSynthetic Sequence 516caggccagtg agagtgtttt
taataatatg ttatcc
3651721DNAArtificialSynthetic Sequence 517gatgcatccg atctggcatc t
2151839DNAArtificialSynthetic Sequence 518gcagggtata aaagtgatag
taatgatggc gataatgtt 3951915DNAArtificialSynthetic Sequence
519aggaattcaa taacc 1552048DNAArtificialSynthetic Sequence
520atcattactg gtagtggtag aacgtactac gcgaactggg caaaaggc
4852130DNAArtificialSynthetic Sequence 521ggccatcctg gtcttggtag
tggtaacatc 30522121PRTArtificialSynthetic Polypeptide 522Met Asp
Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15
Leu Pro Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Ala Ser Ser 20
25 30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ser
Ser 35 40 45 Gln Ser Val Tyr Asn Asn Tyr Leu Ser Trp Tyr Gln Gln
Lys Pro Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Thr Ala Ser
Ser Leu Ala Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Lys Gly Ser Gly
Ser Gly Thr Gln Phe Thr Leu 85 90 95 Thr Ile Ser Glu Val Gln Cys
Asp Asp Ala Ala Thr Tyr Tyr Cys Gln 100 105 110 Gly Tyr Tyr Ser Gly
Pro Ile Ile Thr 115 120 523122PRTArtificialSynthetic Polypeptide
523Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val
Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly
Phe Ser Leu Asn 35 40 45 Asn Tyr Tyr Ile Gln Trp Val Arg Gln Ala
Pro Gly Glu Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Ala Gly
Gly Ser Ala Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Asn Gly Arg Phe Thr
Ile Ala Lys Thr Ser Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr
Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg
Gly Thr Phe Asp Gly Tyr Glu Leu 115 120 52412PRTArtificialSynthetic
Peptide 524Gln Ser Ser Gln Ser Val Tyr Asn Asn Tyr Leu Ser 1 5 10
5257PRTArtificialSynthetic Peptide 525Thr Ala Ser Ser Leu Ala Ser 1
5 52610PRTArtificialSynthetic Peptide 526Gln Gly Tyr Tyr Ser Gly
Pro Ile Ile Thr 1 5 10 5275PRTArtificialSynthetic Peptide 527Asn
Tyr Tyr Ile Gln 1 5 52816PRTArtificialSynthetic Peptide 528Ile Ile
Tyr Ala Gly Gly Ser Ala Tyr Tyr Ala Thr Trp Ala Asn Gly 1 5 10 15
5298PRTArtificialSynthetic Peptide 529Gly Thr Phe Asp Gly Tyr Glu
Leu 1 5 530363DNAArtificialSynthetic Sequence 530atggacacga
gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgcgc
aagtgctgac ccagactgca tcgtccgtgt ctgcagctgt gggaggcaca
120gtcaccatca attgccagtc cagtcagagt gtttataata actacttatc
ctggtatcag 180cagaaaccag ggcagcctcc caagctcctg atctatactg
catccagcct ggcatctggg 240gtcccatcgc ggttcaaagg cagtggatct
gggacacagt tcactctcac catcagcgaa 300gtgcagtgtg acgatgctgc
cacttactac tgtcaaggct attatagtgg tcctataatt 360act
363531366DNAArtificialSynthetic Sequence 531atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagcctctg
gattctccct caataactac tacatacaat gggtccgcca ggctccaggg
180gaggggctgg aatggatcgg gatcatttat gctggtggta gcgcatacta
cgcgacctgg 240gcaaacggcc gattcaccat cgccaaaacc tcgtcgacca
cggtggatct gaagatgacc 300agtctgacaa ccgaggacac ggccacctat
ttctgtgcca gagggacatt tgatggttat 360gagttg
36653236DNAArtificialSynthetic Sequence 532cagtccagtc agagtgttta
taataactac ttatcc 3653321DNAArtificialSynthetic Sequence
533actgcatcca gcctggcatc t 2153430DNAArtificialSynthetic Sequence
534caaggctatt atagtggtcc tataattact 3053515DNAArtificialSynthetic
Sequence 535aactactaca tacaa 1553648DNAArtificialSynthetic Sequence
536atcatttatg ctggtggtag cgcatactac gcgacctggg caaacggc
4853724DNAArtificialSynthetic Sequence 537gggacatttg atggttatga
gttg 24538122PRTArtificialSynthetic Polypeptide 538Met Asp Thr Arg
Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro
Gly Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Pro 20 25 30
Val Ser Val Pro Val Gly Asp Thr Val Thr Ile Ser Cys Gln Ser Ser 35
40 45 Glu Ser Val Tyr Ser Asn Asn Leu Leu Ser Trp Tyr Gln Gln Lys
Pro 50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn
Leu Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser
Gly Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Gly Ala Gln Cys Asp
Asp Ala Ala Thr Tyr Tyr Cys 100 105 110 Gln Gly Tyr Tyr Ser Gly Val
Ile Asn Ser 115 120 539124PRTArtificialSynthetic Polypeptide 539Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser
Leu Ser 35 40 45 Ser Tyr Phe Met Ser Trp Val Arg Gln Ala Pro Gly
Glu Gly Leu Glu 50 55 60 Tyr Ile Gly Phe Ile Asn Pro Gly Gly Ser
Ala Tyr Tyr Ala Ser Trp 65 70 75 80 Ala Ser Gly Arg Leu Thr Ile Ser
Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Ile Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Ile Leu Ile
Val Ser Tyr Gly Ala Phe Thr Ile 115 120 54013PRTArtificialSynthetic
Peptide 540Gln Ser Ser Glu Ser Val Tyr Ser Asn Asn Leu Leu Ser 1 5
10 5417PRTArtificialSynthetic Peptide 541Arg Ala Ser Asn Leu Ala
Ser 1 5 54210PRTArtificialSynthetic Peptide 542Gln Gly Tyr Tyr Ser
Gly Val Ile Asn Ser 1 5 10 5435PRTArtificialSynthetic Peptide
543Ser Tyr Phe Met Ser 1 5 54416PRTArtificialSynthetic Peptide
544Phe Ile Asn Pro Gly Gly Ser Ala Tyr Tyr Ala Ser Trp Ala Ser Gly
1 5 10 15 54511PRTArtificialSynthetic Peptide 545Ile Leu Ile Val
Ser Tyr Gly Ala Phe Thr Ile 1 5 10 546366DNAArtificialSynthetic
Sequence 546atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgccc aagtgctgac ccagactcca tcccctgtgt ctgtccctgt
gggagacaca 120gtcaccatca gttgccagtc cagtgagagc gtttatagta
ataacctctt atcctggtat 180cagcagaaac cagggcagcc tcccaagctc
ctgatctaca gggcatccaa tctggcatct 240ggtgtcccat cgcggttcaa
aggcagtgga tctgggacac agttcactct caccatcagc 300ggcgcacagt
gtgacgatgc tgccacttac tactgtcaag gctattatag tggtgtcatt 360aatagt
366547372DNAArtificialSynthetic Sequence 547atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtgtctg
gattctccct cagtagctac ttcatgagct gggtccgcca ggctccaggg
180gaggggctgg aatacatcgg attcattaat cctggtggta gcgcatacta
cgcgagctgg 240gcgagtggcc gactcaccat ctccaaaacc tcgaccacgg
tagatctgaa aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagga ttcttattgt ttcttatgga 360gcctttacca tc
37254839DNAArtificialSynthetic Sequence 548cagtccagtg agagcgttta
tagtaataac ctcttatcc 3954921DNAArtificialSynthetic Sequence
549agggcatcca atctggcatc t 2155030DNAArtificialSynthetic Sequence
550caaggctatt atagtggtgt cattaatagt 3055115DNAArtificialSynthetic
Sequence 551agctacttca tgagc 1555248DNAArtificialSynthetic Sequence
552ttcattaatc ctggtggtag cgcatactac gcgagctggg cgagtggc
4855333DNAArtificialSynthetic Sequence 553attcttattg tttcttatgg
agcctttacc atc 33554122PRTArtificialSynthetic Polypeptide 554Met
Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser
20 25 30 Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln
Ala Thr 35 40 45 Glu Ser Ile Gly Asn Glu Leu Ser Trp Tyr Gln Gln
Lys Pro Gly Gln 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
Thr Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly
Ser Gly Thr Gln Phe Thr Leu Thr 85 90 95 Ile Thr Gly Val Glu Cys
Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Ser
Ala Asn Ile Asp Asn Ala 115 120 555128PRTArtificialSynthetic
Polypeptide 555Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val
Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr
Val Ser Gly Phe Ser Leu Ser 35 40 45 Lys Tyr Tyr Met Ser Trp Val
Arg Gln Ala Pro Glu Lys Gly Leu Lys 50 55 60 Tyr Ile Gly Tyr Ile
Asp Ser Thr Thr Val Asn Thr Tyr Tyr Ala Thr 65 70 75 80 Trp Ala Arg
Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp 85 90 95 Leu
Lys Ile Thr Ser Pro Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 100 105
110 Ala Arg Gly Ser Thr Tyr Phe Thr Asp Gly Gly His Arg Leu Asp Leu
115 120 125 55611PRTArtificialSynthetic Peptide 556Gln Ala Thr Glu
Ser Ile Gly Asn Glu Leu Ser 1 5 10 5577PRTArtificialSynthetic
Peptide 557Ser Ala Ser Thr Leu Ala Ser 1 5
55812PRTArtificialSynthetic Peptide 558Gln Gln Gly Tyr Ser Ser Ala
Asn Ile Asp Asn Ala 1 5 10 5595PRTArtificialSynthetic Peptide
559Lys Tyr Tyr Met Ser 1 5 56017PRTArtificialSynthetic Peptide
560Tyr Ile Asp Ser Thr Thr Val Asn Thr Tyr Tyr Ala Thr Trp Ala Arg
1 5 10 15 Gly 56114PRTArtificialSynthetic Peptide 561Gly Ser Thr
Tyr Phe Thr Asp Gly Gly His Arg Leu Asp Leu 1 5 10
562366DNAArtificialSynthetic Sequence 562atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac
ccagactcca gcctctgtgg aggtagctgt gggaggcaca 120gtcaccatca
agtgccaggc cactgagagc attggcaatg agttatcctg gtatcagcag
180aaaccagggc aggctcccaa gctcctgatc tattctgcat ccactctggc
atctggggtc 240ccatcgcggt tcaaaggcag tggatctggg acacagttca
ctctcaccat caccggcgtg 300gagtgtgatg atgctgccac ttactactgt
caacagggtt atagtagtgc taatattgat 360aatgct
366563384DNAArtificialSynthetic Sequence 563atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120accgtctctg
gattctccct cagtaagtac tacatgagct gggtccgcca ggctccagag
180aaggggctga aatacatcgg atacattgat agtactactg ttaatacata
ctacgcgacc 240tgggcgagag gccgattcac catctccaaa acctcgacca
cggtggatct gaagatcacc 300agtccgacaa gtgaggacac ggccacctat
ttctgtgcca gaggaagtac ttattttact 360gatggaggcc atcggttgga tctc
38456433DNAArtificialSynthetic Sequence 564caggccactg agagcattgg
caatgagtta tcc 3356521DNAArtificialSynthetic Sequence 565tctgcatcca
ctctggcatc t 2156636DNAArtificialSynthetic Sequence 566caacagggtt
atagtagtgc taatattgat aatgct 3656715DNAArtificialSynthetic Sequence
567aagtactaca tgagc 1556851DNAArtificialSynthetic Sequence
568tacattgata gtactactgt taatacatac tacgcgacct gggcgagagg c
5156942DNAArtificialSynthetic Sequence 569ggaagtactt attttactga
tggaggccat cggttggatc tc 42570122PRTArtificialSynthetic Polypeptide
570Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro
Ala Ser 20 25 30 Val Glu Val Ala Val Gly Gly Thr Val Thr Ile Lys
Cys Gln Ala Thr 35 40 45 Glu Ser Ile Gly Asn Glu Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Gln 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Ser
Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly
Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr 85 90 95 Ile Thr Gly Val
Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr
Ser Ser Ala Asn Ile Asp Asn Ala 115 120
571124PRTArtificialSynthetic Polypeptide 571Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser 35 40 45
Thr Tyr Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50
55 60 Trp Ile Gly Ser Ile Thr Ile Asp Gly Arg Thr Tyr Tyr Ala Ser
Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Val Ser Lys Ser Ser Thr Thr
Val Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Thr Gly Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Ile Leu Ile Val Ser Tyr Gly Ala
Phe Thr Ile 115 120 57211PRTArtificialSynthetic Peptide 572Gln Ala
Thr Glu Ser Ile Gly Asn Glu Leu Ser 1 5 10
5737PRTArtificialSynthetic Peptide 573Ser Ala Ser Thr Leu Ala Ser 1
5 57412PRTArtificialSynthetic Peptide 574Gln Gln Gly Tyr Ser Ser
Ala Asn Ile Asp Asn Ala 1 5 10 5755PRTArtificialSynthetic Peptide
575Thr Tyr Asn Met Gly 1 5 57616PRTArtificialSynthetic Peptide
576Ser Ile Thr Ile Asp Gly Arg Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
1 5 10 15 57711PRTArtificialSynthetic Peptide 577Ile Leu Ile Val
Ser Tyr Gly Ala Phe Thr Ile 1 5 10 578366DNAArtificialSynthetic
Sequence 578atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct atgatatgac ccagactcca gcctctgtgg aggtagctgt
gggaggcaca 120gtcaccatca agtgccaggc cactgagagc attggcaatg
agttatcctg gtatcagcag 180aaaccagggc aggctcccaa gctcctgatc
tattctgcat ccactctggc atctggggtc 240ccatcgcggt tcaaaggcag
tggatctggg acacagttca ctctcaccat caccggcgtg 300gagtgtgatg
atgctgccac ttactactgt caacagggtt atagtagtgc taatattgat 360aatgct
366579372DNAArtificialSynthetic Sequence 579atggagactg
ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggta acgcctggga cacccctgac actcacctgc 120acagtctctg
gattctccct cagtacctac aacatgggct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aagtattact attgatggtc gcacatacta
cgcgagctgg 240gcgaaaggcc gattcaccgt ctccaaaagc tcgaccacgg
tggatctgaa aatgaccagt 300ctgacaaccg gggacacggc cacctatttc
tgtgccagga ttcttattgt ttcttatggg 360gcctttacca tc
37258033DNAArtificialSynthetic Sequence 580caggccactg agagcattgg
caatgagtta tcc 3358121DNAArtificialSynthetic Sequence 581tctgcatcca
ctctggcatc t 2158236DNAArtificialSynthetic Sequence 582caacagggtt
atagtagtgc taatattgat aatgct 3658315DNAArtificialSynthetic Sequence
583acctacaaca tgggc 1558448DNAArtificialSynthetic Sequence
584agtattacta ttgatggtcg cacatactac gcgagctggg cgaaaggc
4858533DNAArtificialSynthetic Sequence 585attcttattg tttcttatgg
ggcctttacc atc 33586105PRTArtificialSynthetic Polypeptide 586Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 1 5 10
15 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
20 25 30 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly 35 40 45 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr 50 55 60 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His 65 70 75 80 Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val 85 90 95 Thr Lys Ser Phe Asn Arg
Gly Glu Cys 100 105 587315DNAArtificialSynthetic Sequence
587gtggctgcac catctgtctt catcttcccg ccatctgatg agcagttgaa
atctggaact 60gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt
acagtggaag 120gtggataacg ccctccaatc gggtaactcc caggagagtg
tcacagagca ggacagcaag 180gacagcacct acagcctcag cagcaccctg
acgctgagca aagcagacta cgagaaacac 240aaagtctacg cctgcgaagt
cacccatcag ggcctgagct cgcccgtcac aaagagcttc 300aacaggggag agtgt
315588330PRTArtificialSynthetic Polypeptide 588Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
589990DNAArtificialSynthetic Sequence 589gcctccacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagag agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacgcc 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctac agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc
tccgggtaaa 99059015PRTArtificialSynthetic Peptide 590Val Pro Pro
Gly Glu Asp Ser Lys Asp Val Ala Ala Pro His Arg 1 5 10 15
59115PRTArtificialSynthetic Peptide 591Gly Glu Asp Ser Lys Asp Val
Ala Ala Pro His Arg Gln Pro Leu 1 5 10 15
59215PRTArtificialSynthetic Peptide 592Ser Lys Asp Val Ala Ala Pro
His Arg Gln Pro Leu Thr Ser Ser 1 5 10 15
59315PRTArtificialSynthetic Peptide 593Val Ala Ala Pro His Arg Gln
Pro Leu Thr Ser Ser Glu Arg Ile 1 5 10 15
59415PRTArtificialSynthetic Peptide 594Pro His Arg Gln Pro Leu Thr
Ser Ser Glu Arg Ile Asp Lys Gln 1 5 10 15
59515PRTArtificialSynthetic Peptide 595Gln Pro Leu Thr Ser Ser Glu
Arg Ile Asp Lys Gln Ile Arg Tyr 1 5 10 15
59615PRTArtificialSynthetic Peptide 596Thr Ser Ser Glu Arg Ile Asp
Lys Gln Ile Arg Tyr Ile Leu Asp 1 5 10 15
59715PRTArtificialSynthetic Pepide 597Glu Arg Ile Asp Lys Gln Ile
Arg Tyr Ile Leu Asp Gly Ile Ser 1 5 10 15
59815PRTArtificialSynthetic Peptide 598Asp Lys Gln Ile Arg Tyr Ile
Leu Asp Gly Ile Ser Ala Leu Arg 1 5 10 15
59915PRTArtificialSynthetic Peptide 599Ile Arg Tyr Ile Leu Asp Gly
Ile Ser Ala Leu Arg Lys Glu Thr 1 5 10 15
60015PRTArtificialSynthetic Peptide 600Ile Leu Asp Gly Ile Ser Ala
Leu Arg Lys Glu Thr Cys Asn Lys 1 5 10 15
60115PRTArtificialSynthetic Peptide 601Gly Ile Ser Ala Leu Arg Lys
Glu Thr Cys Asn Lys Ser Asn Met 1 5 10 15
60215PRTArtificialSynthetic Peptide 602Ala Leu Arg Lys Glu Thr Cys
Asn Lys Ser Asn Met Cys Glu Ser 1 5 10 15
60315PRTArtificialSynthetic Peptide 603Lys Glu Thr Cys Asn Lys Ser
Asn Met Cys Glu Ser Ser Lys Glu 1 5 10 15
60415PRTArtificialSynthetic Peptide 604Cys Asn Lys Ser Asn Met Cys
Glu Ser Ser Lys Glu Ala Leu Ala 1 5 10 15
60515PRTArtificialSynthetic Peptide 605Ser Asn Met Cys Glu Ser Ser
Lys Glu Ala Leu Ala Glu Asn Asn 1 5 10 15
60615PRTArtificialSynthetic Peptide 606Cys Glu Ser Ser Lys Glu Ala
Leu Ala Glu Asn Asn Leu Asn Leu 1 5 10 15
60715PRTArtificialSynthetic Peptide 607Ser Lys Glu Ala Leu Ala Glu
Asn Asn Leu Asn Leu Pro Lys Met 1 5 10 15
60815PRTArtificialSynthetic Peptide 608Ala Leu Ala Glu Asn Asn Leu
Asn Leu Pro Lys Met Ala Glu Lys 1 5 10 15
60915PRTArtificialSynthetic Peptide 609Glu Asn Asn Leu Asn Leu Pro
Lys Met Ala Glu Lys Asp Gly Cys 1 5 10 15
61015PRTArtificialSynthetic Peptide 610Leu Asn Leu Pro Lys Met Ala
Glu Lys Asp Gly Cys Phe Gln Ser 1 5 10 15
61115PRTArtificialSynthetic Peptide 611Pro Lys Met Ala Glu Lys Asp
Gly Cys Phe Gln Ser Gly Phe Asn 1 5 10 15
61215PRTArtificialSynthetic Peptide 612Ala Glu Lys Asp Gly Cys Phe
Gln Ser Gly Phe Asn Glu Glu Thr 1 5 10 15
61315PRTArtificialSynthetic Peptide 613Asp Gly Cys Phe Gln Ser Gly
Phe Asn Glu Glu Thr Cys Leu Val 1 5 10 15
61415PRTArtificialSynthetic Peptide 614Phe Gln Ser Gly Phe Asn Glu
Glu Thr Cys Leu Val Lys Ile Ile 1 5 10 15
61515PRTArtificialSynthetic Peptide 615Gly Phe Asn Glu Glu Thr Cys
Leu Val Lys Ile Ile Thr Gly Leu 1 5 10 15
61615PRTArtificialSynthetic Peptide 616Glu Glu Thr Cys Leu Val Lys
Ile Ile Thr Gly Leu Leu Glu Phe 1 5 10 15
61715PRTArtificialSynthetic Peptide 617Cys Leu Val Lys Ile Ile Thr
Gly Leu Leu Glu Phe Glu Val Tyr 1 5 10 15
61815PRTArtificialSynthetic Peptide 618Lys Ile Ile Thr Gly Leu Leu
Glu Phe Glu Val Tyr Leu Glu Tyr 1 5 10 15
61915PRTArtificialSynthetic Peptide 619Thr Gly Leu Leu Glu Phe Glu
Val Tyr Leu Glu Tyr Leu Gln Asn 1 5 10 15
62015PRTArtificialSynthetic Peptide 620Leu Glu Phe Glu Val Tyr Leu
Glu Tyr Leu Gln Asn Arg Phe Glu 1 5 10 15
62115PRTArtificialSynthetic Peptide 621Glu Val Tyr Leu Glu Tyr Leu
Gln Asn Arg Phe Glu Ser Ser Glu 1 5 10 15
62215PRTArtificialSynthetic Peptide 622Leu Glu Tyr Leu Gln Asn Arg
Phe Glu Ser Ser Glu Glu Gln Ala 1 5 10 15
62315PRTArtificialSynthetic Peptide 623Leu Gln Asn Arg Phe Glu Ser
Ser Glu Glu Gln Ala Arg Ala Val 1 5 10 15
62415PRTArtificialSynthetic Peptide 624Arg Phe Glu Ser Ser Glu Glu
Gln Ala Arg Ala Val Gln Met Ser 1 5 10 15
62515PRTArtificialSynthetic Peptide 625Ser Ser Glu Glu Gln Ala Arg
Ala Val Gln Met Ser Thr Lys Val 1 5 10 15
62615PRTArtificialSynthetic Peptide 626Glu Gln Ala Arg Ala Val Gln
Met Ser Thr Lys Val Leu Ile Gln 1 5 10 15
62715PRTArtificialSynthetic Peptide 627Arg Ala Val Gln Met Ser Thr
Lys Val Leu Ile Gln Phe Leu Gln 1 5 10 15
62815PRTArtificialSynthetic Peptide 628Gln Met Ser Thr Lys Val Leu
Ile Gln Phe Leu Gln Lys Lys Ala 1 5 10 15
62915PRTArtificialSynthetic Peptide 629Thr Lys Val Leu Ile Gln Phe
Leu Gln Lys Lys Ala Lys Asn Leu 1 5 10 15
63015PRTArtificialSynthetic Peptide 630Leu Ile Gln Phe Leu Gln Lys
Lys Ala Lys Asn Leu Asp Ala Ile 1 5 10 15
63115PRTArtificialSynthetic Peptide 631Phe Leu Gln Lys Lys Ala Lys
Asn Leu Asp Ala Ile Thr Thr Pro 1 5 10 15
63215PRTArtificialSynthetic Peptide 632Lys Lys Ala Lys Asn Leu Asp
Ala Ile Thr Thr Pro Asp Pro Thr 1 5 10 15
63315PRTArtificialSynthetic Peptide 633Lys Asn Leu Asp Ala Ile Thr
Thr Pro Asp Pro Thr Thr Asn Ala 1 5 10 15
63415PRTArtificialSynthetic Peptide 634Asp Ala Ile Thr Thr Pro Asp
Pro Thr Thr Asn Ala Ser Leu Leu 1 5 10 15
63515PRTArtificialSynthetic Peptide 635Thr Thr Pro Asp Pro Thr Thr
Asn Ala Ser Leu Leu Thr Lys Leu 1 5 10 15
63615PRTArtificialSynthetic Peptide 636Asp Pro Thr Thr Asn Ala Ser
Leu Leu Thr Lys Leu Gln Ala Gln 1 5 10 15
63715PRTArtificialSynthetic Peptide 637Thr Asn Ala Ser Leu Leu Thr
Lys Leu Gln Ala Gln Asn Gln Trp 1 5 10 15
63815PRTArtificialSynthetic Peptide 638Ser Leu Leu Thr Lys Leu Gln
Ala Gln Asn Gln Trp Leu Gln Asp 1 5 10 15
63915PRTArtificialSynthetic Peptide 639Thr Lys Leu Gln Ala Gln Asn
Gln Trp Leu Gln Asp Met Thr Thr 1 5 10 15
64015PRTArtificialSynthetic Peptide 640Gln Ala Gln Asn Gln Trp Leu
Gln Asp Met Thr Thr His Leu Ile 1 5 10 15
64115PRTArtificialSynthetic Ppeptide 641Asn Gln Trp Leu Gln Asp Met
Thr Thr His Leu Ile Leu Arg Ser 1 5 10 15
64215PRTArtificialSynthetic Peptide 642Leu Gln Asp Met Thr Thr His
Leu Ile Leu Arg Ser Phe Lys Glu 1 5 10 15
64315PRTArtificialSynthetic Peptide 643Met Thr Thr His Leu Ile Leu
Arg Ser Phe Lys Glu Phe Leu Gln 1 5 10 15
64415PRTArtificialSynthetic Peptide 644His Leu Ile Leu Arg Ser Phe
Lys Glu Phe Leu Gln Ser Ser Leu 1 5 10 15
64515PRTArtificialSynthetic Peptide 645Leu Arg Ser Phe Lys Glu Phe
Leu Gln Ser Ser Leu Arg Ala Leu 1 5 10 15
64615PRTArtificialSynthetic Peptide 646Phe Lys Glu Phe Leu Gln Ser
Ser Leu Arg Ala Leu Arg Gln Met 1 5 10 15
647111PRTArtificialSynthetic Polypeptide 647Ala Tyr Asp Met Thr Gln
Thr Pro Ala Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr
Ile Lys Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu Ser
Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys Leu Leu Ile 35 40 45
Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu
Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser
Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu Val
Val Val Lys Arg 100 105 110 64888PRTArtificialSynthetic Polypeptide
648Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys 85 64988PRTArtificialSynthetic Polypeptide 649Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65
70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys 85
65088PRTArtificialSynthetic Polypeptide 650Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys 85
651111PRTArtificialSynthetic Polypeptide 651Ala Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu Ser
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser
Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105 110 652117PRTArtificialSynthetic
Polypeptide 652Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
Leu Ser Asn Tyr Tyr 20 25 30 Val Thr Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Tyr Gly Ser Asp Glu
Thr Ala Tyr Ala Thr Trp Ala Ile Gly 50 55 60 Arg Phe Thr Ile Ser
Lys Thr Ser Thr Thr Val Asp Leu Lys Met Thr 65 70 75 80 Ser Leu Thr
Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Asp Asp 85 90 95 Ser
Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 65397PRTArtificialSynthetic Polypeptide
653Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser
Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
65497PRTArtificialSynthetic Polypeptide 654Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg 65598PRTArtificialSynthetic Polypeptide
655Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Ser Thr
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys
656120PRTArtificialSynthetic Polypeptide 656Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr Val
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp Ala Ile 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe
Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
120 657120PRTArtificialSynthetic Polypeptide 657Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr
Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
115 120 658166PRTArtificialSynthetic Polypeptide 658Met Glu Thr Gly
Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln
Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30
Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35
40 45 Asn Tyr Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr
Ala Thr Ser 65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Lys Thr Ser
Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Ala Ala Asp
Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Asp Ser Ser Asp Trp
Asp Ala Lys Phe Asn Leu Trp Gly Gln 115 120 125 Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 130 135 140 Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 145 150 155 160
Leu Gly Cys Leu Val Lys 165 65916PRTArtificialSynthetic Peptide
659Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile Gly
1 5 10 15 660122PRTArtificialSynthetic Polypeptide 660Met Asp Thr
Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu
Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25
30 Val Ser Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser
35 40 45 Gln Ser Ile Asn Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro
Gly Gln 50 55 60 Arg Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu
Ala Ser Gly Val 65 70 75 80 Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp
Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Leu Arg Asn
Ile Asp Asn Ala 115 120 661125PRTArtificialSynthetic Polypeptide
661Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val
Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly
Phe Ser Leu Ser 35 40 45 Asn Tyr Tyr Val Thr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Gly Ser
Asp Glu Thr Ala Tyr Ala Thr Trp 65 70 75 80 Ala Ile Gly Arg Phe Thr
Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser
Leu Thr Ala Ala Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Asp
Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu 115 120 125
662366DNAArtificialSynthetic Sequence 662atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac
ccagactcca gcctcggtgt ctgcagctgt gggaggcaca 120gtcaccatca
agtgccaggc cagtcagagc attaacaatg aattatcctg gtatcagcag
180aaaccagggc agcgtcccaa gctcctgatc tatagggcat ccactctggc
atctggggtc 240tcatcgcggt tcaaaggcag tggatctggg acagagttca
ctctcaccat cagcgacctg 300gagtgtgccg atgctgccac ttactactgt
caacagggtt atagtctgag gaatattgat 360aatgct
366663375DNAArtificialSynthetic Sequence 663atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtaactac tacgtgacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatcatttat ggtagtgatg aaacggccta
cgcgacctgg 240gcgataggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ctgacagccg cggacacggc cacctatttc
tgtgccagag atgatagtag tgactgggat 360gcaaaattta acttg
375664450PRTArtificialSynthetic Polypeptide 664Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr
Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Trp Ala Ile
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 445 Gly Lys 450 665450PRTArtificialSynthetic
Polypeptide 665Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Leu Ser Asn Tyr 20 25 30 Tyr Val Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ile Ile Tyr Gly Ser Asp
Glu Thr Ala Tyr Ala Thr Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230
235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445 Gly Lys 450 666216PRTArtificialSynthetic Polypeptide 666Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 1 5 10 15
Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu Leu 20
25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr 35 40 45 Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gly Tyr Ser Leu Arg Asn Ile 85 90 95 Asp Asn Ala Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150
155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210
215 667122PRTArtificialSynthetic Polypeptide 667Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys Ala Tyr Asp Met Thr Gln Thr Pro Ala Ser 20 25 30 Val
Glu Val Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40
45 Glu Thr Ile Tyr Ser Trp Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln
50 55 60 Pro Pro Lys Leu Leu Ile Tyr Gln Ala Ser Asp Leu Ala Ser
Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ala Gly Thr Glu
Tyr Thr Leu Thr 85 90 95 Ile Ser Gly Val Gln Cys Asp Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln 100 105 110 Gly Tyr Ser Gly Ser Asn Val Asp
Asn Val 115 120 668126PRTArtificialSynthetic Polypeptide 668Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Arg Leu Val Thr 20
25 30 Pro Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
Leu 35 40 45 Asn Asp His Ala Met Gly Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 50 55 60 Glu Tyr Ile Gly Phe Ile Asn Ser Gly Gly Ser
Ala Arg Tyr Ala Ser 65 70 75 80 Trp Ala Glu Gly Arg Phe Thr Ile Ser
Arg Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Leu Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Val Arg Gly Gly Ala
Val Trp Ser Ile His Ser Phe Asp Pro 115 120 125
669366DNAArtificialSynthetic Sequence 669atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60agatgtgcct atgatatgac
ccagactcca gcctctgtgg aggtagctgt gggaggcaca 120gtcaccatca
attgccaggc cagtgagacc atttacagtt ggttatcctg gtatcagcag
180aagccagggc agcctcccaa gctcctgatc taccaggcat ccgatctggc
atctggggtc 240ccatcgcgat tcagcggcag tggggctggg acagagtaca
ctctcaccat cagcggcgtg 300cagtgtgacg atgctgccac ttactactgt
caacagggtt atagtggtag taatgttgat 360aatgtt
366670378DNAArtificialSynthetic Sequence 670atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60gagcagctga aggagtccgg
gggtcgcctg gtcacgcctg ggacacccct gacacttacc 120tgcacagcct
ctggattctc cctcaatgac catgcaatgg gctgggtccg ccaggctcca
180gggaaggggc tggaatacat cggattcatt aatagtggtg gtagcgcacg
ctacgcgagc 240tgggcagaag gccgattcac catctccaga acctcgacca
cggtggatct gaaaatgacc 300agtctgacaa ccgaggacac ggccacctat
ttctgtgtca gagggggtgc tgtttggagt 360attcatagtt ttgatccc
378671123PRTArtificialSynthetic Polypeptide 671Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser 35 40
45 Gln Ser Val Tyr Asp Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro
50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu
Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Val Gly Ser Gly Ser Gly
Thr Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val Gln Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Tyr Asp Asp Asp Ser
Asp Asn Ala 115 120 672125PRTArtificialSynthetic Polypeptide 672Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
Leu Ser 35 40 45 Val Tyr Tyr Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu 50 55 60 Trp Ile Gly Phe Ile Thr Met Ser Asp Asn
Ile Asn Tyr Ala Ser Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser
Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Lys Met Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Ser Arg Gly
Trp Gly Thr Met Gly Arg Leu Asp Leu 115 120 125
673369DNAArtificialSynthetic Sequence 673atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgccg ccgtgctgac
ccagactcca tctcccgtgt ctgcagctgt gggaggcaca 120gtcagcatca
gttgccaggc cagtcagagt gtttatgaca acaactactt atcctggttt
180cagcagaaac cagggcagcc tcccaagctc ctgatctatg gtgcatccac
tctggcatct 240ggggtcccat cgcggttcgt gggcagtgga tctgggacac
agttcactct caccatcaca 300gacgtgcagt gtgacgatgc tgccacttac
tattgtgcag gcgtttatga tgatgatagt 360gataatgcc
369674375DNAArtificialSynthetic Sequence 674atggagactg ggctgcgctg
gcttctcctg gtggctgtgc tcaaaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acccctggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtgtctac tacatgaact gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg attcattaca atgagtgata atataaatta
cgcgagctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagga gtcgtggctg gggtacaatg 360ggtcggttgg atctc
375675123PRTArtificialSynthetic Polypeptide 675Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Ile Cys Asp Pro Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val
Ser Ala Pro Val Gly Gly Thr Val Ser Ile Ser Cys Gln Ala Ser 35 40
45 Gln Ser Val Tyr Glu Asn Asn Tyr Leu Ser Trp Phe Gln Gln Lys Pro
50 55 60 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Leu
Asp Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly
Thr Gln Phe Thr 85 90 95 Leu Thr Ile Thr Asp Val Gln Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Val Tyr Asp Asp Asp Ser
Asp Asp Ala 115 120 676126PRTArtificialSynthetic Polypeptide 676Met
Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10
15 Val Gln Cys Gln Glu Gln Leu Lys Glu Ser Gly Gly Gly Leu Val Thr
20 25 30 Pro Gly Gly Thr Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe
Ser Leu 35 40 45 Asn Ala Tyr Tyr Met Asn Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Phe Ile Thr Leu Asn Asn
Asn Val Ala Tyr Ala Asn 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Phe
Ser Lys Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Met Thr Ser Pro
Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Ser Arg
Gly Trp Gly Ala Met Gly Arg Leu Asp Leu 115 120 125
677369DNAArtificialSynthetic Sequence 677atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60atatgtgacc ctgtgctgac
ccagactcca tctcccgtat ctgcacctgt gggaggcaca 120gtcagcatca
gttgccaggc cagtcagagt gtttatgaga acaactattt atcctggttt
180cagcagaaac cagggcagcc tcccaagctc ctgatctatg gtgcatccac
tctggattct 240ggggtcccat cgcggttcaa aggcagtgga tctgggacac
agttcactct caccattaca 300gacgtgcagt gtgacgatgc tgccacttac
tattgtgcag gcgtttatga tgatgatagt 360gatgatgcc
369678378DNAArtificialSynthetic Sequence 678atggagactg ggctgcgctg
gcttctcctg gtggctgtgc tcaaaggtgt ccagtgtcag 60gagcagctga aggagtccgg
aggaggcctg gtaacgcctg gaggaaccct gacactcacc 120tgcacagcct
ctggattctc cctcaatgcc tactacatga actgggtccg ccaggctcca
180gggaaggggc tggaatggat cggattcatt actctgaata ataatgtagc
ttacgcgaac 240tgggcgaaag gccgattcac cttctccaaa acctcgacca
cggtggatct gaaaatgacc 300agtccgacac ccgaggacac ggccacctat
ttctgtgcca ggagtcgtgg ctggggtgca 360atgggtcggt tggatctc
378679122PRTArtificialSynthetic Polypeptide 679Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Thr Phe Ala Gln Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val
Ser Ala Ala Val Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser 35 40
45 Gln Ser Val Asp Asp Asn Asn Trp Leu Gly Trp Tyr Gln Gln Lys Arg
50 55 60 Gly Gln Pro Pro Lys Tyr Leu Ile Tyr Ser Ala Ser Thr Leu
Ala Ser 65 70 75 80 Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly
Thr Gln Phe Thr 85 90 95 Leu Thr Ile Ser Asp Leu Glu Cys Asp Asp
Ala Ala Thr Tyr Tyr Cys 100 105 110 Ala Gly Gly Phe Ser Gly Asn Ile
Phe Ala 115 120 680122PRTArtificialSynthetic Polypeptide 680Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20
25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu
Ser 35 40 45 Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Gly Gly Phe Gly Thr Thr
Tyr Tyr Ala Thr Trp 65 70 75 80 Ala Lys Gly Arg Phe Thr Ile Ser Lys
Thr Ser Thr Thr Val Asp Leu 85 90 95 Arg Ile Thr Ser Pro Thr Thr
Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Gly Gly Pro Gly
Asn Gly Gly Asp Ile 115 120 681366DNAArtificialSynthetic Sequence
681atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60acatttgccc aagtgctgac ccagactcca tcgcctgtgt ctgcagctgt
gggaggcaca 120gtcaccatca actgccaggc cagtcagagt gttgatgata
acaactggtt aggctggtat 180cagcagaaac gagggcagcc tcccaagtac
ctgatctatt ctgcatccac tctggcatct 240ggggtcccat cgcggttcaa
aggcagtgga tctgggacac agttcactct caccatcagc 300gacctggagt
gtgacgatgc tgccacttac tactgtgcag gcggttttag tggtaatatc 360tttgct
366682366DNAArtificialSynthetic Sequence 682atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagtctctg
gcttctccct cagtagctat gcaatgagct gggtccgcca ggctccagga
180aaggggctgg agtggatcgg aatcattggt ggttttggta ccacatacta
cgcgacctgg 240gcgaaaggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgag aatcaccagt 300ccgacaaccg aggacacggc cacctatttc
tgtgccagag gtggtcctgg taatggtggt 360gacatc
366683122PRTArtificialSynthetic Polypeptide 683Met Asp Thr Arg Ala
Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Thr Phe Ala Ala Val Leu Thr Gln Thr Pro Ser Pro 20 25 30 Val
Ser Val Pro Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ser Ser 35 40
45 Gln Ser Val Tyr Asn Asn Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly
50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Gln Ala Ser Lys Leu Ala
Ser Gly 65 70 75 80 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Gln Phe Thr Leu 85 90 95 Thr Ile Ser Gly Val Gln Cys Asp Asp Ala
Ala Thr Tyr Tyr Cys Leu 100 105 110 Gly Gly Tyr Asp Asp Asp Ala Asp
Asn Ala 115 120 684128PRTArtificialSynthetic Polypeptide 684Met Glu
Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15
Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20
25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu
Ser 35 40 45 Asp Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Ala Gly Ser Gly Ser
Thr Trp Tyr Ala Ser 65 70 75 80 Trp Ala Lys Gly Arg Phe Thr Ile Ser
Lys Thr Ser Thr Thr Val Asp 85 90 95 Leu Lys Ile Thr Ser Pro Thr
Thr Glu Asp Thr Ala Thr Tyr Phe Cys 100 105 110 Ala Arg Asp Gly Tyr
Asp Asp Tyr Gly Asp Phe Asp Arg Leu Asp Leu 115 120 125
685366DNAArtificialSynthetic Sequence 685atggacacga gggcccccac
tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60acatttgcag ccgtgctgac
ccagacacca tcgcccgtgt ctgtacctgt gggaggcaca 120gtcaccatca
agtgccagtc cagtcagagt gtttataata atttcttatc gtggtatcag
180cagaaaccag ggcagcctcc caagctcctg atctaccagg catccaaact
ggcatctggg 240gtcccagata ggttcagcgg cagtggatct gggacacagt
tcactctcac catcagcggc 300gtgcagtgtg acgatgctgc cacttactac
tgtctaggcg gttatgatga tgatgctgat 360aatgct
366686384DNAArtificialSynthetic Sequence 686atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60tcggtggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac
gctcacctgc 120acagtctctg gaatcgacct cagtgactat gcaatgagct
gggtccgcca ggctccaggg 180aaggggctgg aatggatcgg aatcatttat
gctggtagtg gtagcacatg gtacgcgagc 240tgggcgaaag gccgattcac
catctccaaa acctcgacca cggtggatct gaaaatcacc 300agtccgacaa
ccgaggacac ggccacctat ttctgtgcca gagatggata cgatgactat
360ggtgatttcg atcgattgga tctc 384687122PRTArtificialSynthetic
Polypeptide 687Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu
Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Tyr Asp Met Thr
Gln Thr Pro Ala Ser 20 25 30 Val Ser Ala Ala Val Gly Gly Thr Val
Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln Ser Ile Asn Asn Glu Leu
Ser Trp Tyr Gln Gln Lys Ser Gly Gln 50 55 60 Arg Pro Lys Leu Leu
Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val 65 70 75 80 Ser Ser Arg
Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile
Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 100 105
110 Gly Tyr Ser Leu Arg Asn Ile Asp Asn Ala 115 120
688125PRTArtificialSynthetic Polypeptide 688Met Glu Thr Gly Leu Arg
Trp Leu Leu Leu Val Ala Val Leu Ser Gly 1 5 10 15 Val Gln Cys Gln
Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr
Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40 45
Asn Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50
55 60 Trp Ile Gly Met Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Asn
Trp 65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr
Val Asp Leu 85 90 95 Lys Met Thr Ser Leu Thr Ala Ala Asp Thr Ala
Thr Tyr Phe Cys Ala 100 105 110 Arg Asp Asp Ser Ser Asp Trp Asp Ala
Lys Phe Asn Leu 115 120 125 689366DNAArtificialSynthetic Sequence
689atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct
cccaggtgcc 60agatgtgcct atgatatgac ccagactcca gcctcggtgt ctgcagctgt
gggaggcaca 120gtcaccatca aatgccaggc cagtcagagc attaacaatg
aattatcctg gtatcagcag 180aaatcagggc agcgtcccaa gctcctgatc
tatagggcat ccactctggc atctggggtc 240tcatcgcggt tcaaaggcag
tggatctggg acagagttca ctctcaccat cagcgacctg 300gagtgtgccg
atgctgccac ttactactgt caacagggtt atagtctgag gaatattgat 360aatgct
366690375DNAArtificialSynthetic Sequence 690atggagactg ggctgcgctg
gcttctcctg gtcgctgtgc tctcaggtgt ccagtgtcag 60tcgctggagg agtccggggg
tcgcctggtc acgcctggga cacccctgac actcacctgc 120acagcctctg
gattctccct cagtaactac tacatgacct gggtccgcca ggctccaggg
180aaggggctgg aatggatcgg aatgatttat ggtagtgatg aaacagccta
cgcgaactgg 240gcgataggcc gattcaccat ctccaaaacc tcgaccacgg
tggatctgaa aatgaccagt 300ctgacagccg cggacacggc cacctatttc
tgtgccagag atgatagtag tgactgggat 360gcaaaattta acttg
375691450PRTArtificialSynthetic Polypeptide 691Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr
Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Met Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Asn Trp Ala Ile
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 445 Gly Lys 450 692450PRTArtificialSynthetic
Polypeptide 692Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Ser Leu Ser Asn Tyr 20 25 30 Tyr Met Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Met Ile Tyr Gly Ser Asp
Glu Thr Ala Tyr Ala Asn Ser Ala Ile 50 55 60 Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
Asp Asp Ser Ser Asp Trp Asp Ala Lys Phe Asn Leu Trp Gly Gln 100 105
110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230
235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355
360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
693217PRTArtificialSynthetic Polypeptide 693Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu Ser
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser
Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180
185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
69433DNAArtificialSynthetic Sequence 694caggccagtc agagcattaa
caatgagtta tcc 3369536DNAArtificialSynthetic Sequence 695caacagggtt
atagtctgag gaacattgat aatgct 3669648DNAArtificialSynthetic Sequence
696atcatctatg gtagtgatga aaccgcctac gctacctccg ctataggc
4869736DNAArtificialSynthetic Sequence 697gatgatagta gtgactggga
tgcaaagttc aacttg 36698336DNAArtificialSynthetic Sequence
698gctatccaga tgacccagtc tccttcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc aggccagtca gagcattaac aatgagttat cctggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagg gcatccactc
tggcatctgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagac
ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta
ctgccaacag ggttatagtc tgaggaacat tgataatgct 300ttcggcggag
ggaccaaggt ggaaatcaaa cgtacg 336699112PRTArtificialSynthetic
Polypeptide 699Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln
Ser Ile Asn Asn Glu 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Ser Thr Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Leu Arg Asn 85 90 95 Ile
Asp Asn Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105
110 700360DNAArtificialSynthetic Sequence 700gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
ctccctcagt aactactacg tgacctgggt ccgtcaggct 120ccagggaagg
ggctggagtg ggtcggcatc atctatggta gtgatgaaac cgcctacgct
180acctccgcta taggccgatt caccatctcc agagacaatt ccaagaacac
cctgtatctt 240caaatgaaca gcctgagagc tgaggacact gctgtgtatt
actgtgctag agatgatagt 300agtgactggg atgcaaagtt caacttgtgg
ggccaaggga ccctcgtcac cgtctcgagc 360701651DNAArtificialSynthetic
Sequence 701gctatccaga tgacccagtc tccttcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc aggccagtca gagcattaac aatgagttat cctggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatagg gcatccactc
tggcatctgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagac
ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta
ctgccaacag ggttatagtc tgaggaacat tgataatgct 300ttcggcggag
ggaccaaggt ggaaatcaaa cgtacggtgg ctgcaccatc tgtcttcatc
360ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg
cctgctgaat 420aacttctatc ccagagaggc caaagtacag tggaaggtgg
ataacgccct ccaatcgggt 480aactcccagg agagtgtcac agagcaggac
agcaaggaca gcacctacag cctcagcagc 540accctgacgc tgagcaaagc
agactacgag aaacacaaag tctacgcctg cgaagtcacc 600catcagggcc
tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t
651702217PRTArtificialSynthetic Polypeptide 702Ala Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu
Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr
Ser Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170
175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
7031350DNAArtificialSynthetic Sequence 703gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
ctccctcagt aactactacg tgacctgggt ccgtcaggct 120ccagggaagg
ggctggagtg ggtcggcatc atctatggta gtgatgaaac cgcctacgct
180acctccgcta taggccgatt caccatctcc agagacaatt ccaagaacac
cctgtatctt 240caaatgaaca gcctgagagc tgaggacact gctgtgtatt
actgtgctag agatgatagt 300agtgactggg atgcaaagtt caacttgtgg
ggccaaggga ccctcgtcac cgtctcgagc 360gcctccacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 420ggcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagag agttgagccc 660aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
ctgaggtcaa gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc cgcgggagga gcagtacgcc 900agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1320cagaagagcc tctccctgtc tccgggtaaa
1350704450PRTArtificialSynthetic Polypeptide 704Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Tyr
Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala Thr Ser Ala Ile
50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95 Arg Asp Asp Ser Ser Asp Trp Asp Ala Lys
Phe Asn Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 445 Gly Lys 450 705705DNAArtificialSynthetic Sequence
705atgaagtggg taacctttat ttcccttctg tttctcttta gcagcgctta
ttccgctatc 60cagatgaccc agtctccttc ctccctgtct gcatctgtag gagacagagt
caccatcact 120tgccaggcca gtcagagcat taacaatgag ttatcctggt
atcagcagaa accagggaaa 180gcccctaagc tcctgatcta tagggcatcc
actctggcat ctggggtccc atcaaggttc 240agcggcagtg gatctgggac
agacttcact ctcaccatca gcagcctgca gcctgatgat 300tttgcaactt
attactgcca acagggttat agtctgagga acattgataa tgctttcggc
360ggagggacca aggtggaaat caaacgtacg gtggctgcac catctgtctt
catcttcccg 420ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 480tatcccagag aggccaaagt acagtggaag
gtggataacg ccctccaatc gggtaactcc 540caggagagtg tcacagagca
ggacagcaag gacagcacct acagcctcag cagcaccctg 600acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag
660ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt
705706235PRTArtificialSynthetic Polypeptide 706Met Lys Trp Val Thr
Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 20 25 30 Val
Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn 35 40
45 Asn Glu Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
50 55 60 Leu Ile Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser
Arg Phe 65 70 75 80 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu 85 90 95 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly Tyr Ser Leu 100 105 110 Arg Asn Ile Asp Asn Ala Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 165 170
175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 225 230 235 7071404DNAArtificialSynthetic Sequence
707atgaagtggg taacctttat ttcccttctg tttctcttta gcagcgctta
ttccgaggtg 60cagctggtgg agtctggggg aggcttggtc cagcctgggg ggtccctgag
actctcctgt 120gcagcctctg gattctccct cagtaactac tacgtgacct
gggtccgtca ggctccaggg 180aaggggctgg agtgggtcgg catcatctat
ggtagtgatg aaaccgccta cgctacctcc 240gctataggcc gattcaccat
ctccagagac aattccaaga acaccctgta tcttcaaatg 300aacagcctga
gagctgagga cactgctgtg tattactgtg ctagagatga tagtagtgac
360tgggatgcaa agttcaactt gtggggccaa gggaccctcg tcaccgtctc
gagcgcctcc 420accaagggcc catcggtctt ccccctggca ccctcctcca
agagcacctc tgggggcaca 480gcggccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg
cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600tactccctca
gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc
660tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agagagttga
gcccaaatct 720tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg
aactcctggg gggaccgtca 780gtcttcctct tccccccaaa acccaaggac
accctcatga tctcccggac ccctgaggtc 840acatgcgtgg tggtggacgt
gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900gacggcgtgg
aggtgcataa tgccaagaca aagccgcggg aggagcagta cgccagcacg
960taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg
caaggagtac 1020aagtgcaagg tctccaacaa agccctccca gcccccatcg
agaaaaccat ctccaaagcc 1080aaagggcagc cccgagaacc acaggtgtac
accctgcccc catcccggga ggagatgacc 1140aagaaccagg tcagcctgac
ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1200gagtgggaga
gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac
1260tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag
gtggcagcag 1320gggaacgtct tctcatgctc cgtgatgcat gaggctctgc
acaaccacta cacgcagaag 1380agcctctccc tgtctccggg taaa
1404708468PRTArtificialSynthetic Polypeptide 708Met Lys Trp Val Thr
Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser 35 40
45 Asn Tyr Tyr Val Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60 Trp Val Gly Ile Ile Tyr Gly Ser Asp Glu Thr Ala Tyr Ala
Thr Ser 65 70 75 80 Ala Ile Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu 85 90 95 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr 100 105 110 Cys Ala Arg Asp Asp Ser Ser Asp
Trp Asp Ala Lys Phe Asn Leu Trp 115 120 125 Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170
175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295
300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr
305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420
425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465
709111PRTArtificialSynthetic Polypeptide 709Ala Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Ser Ile Asn Asn Glu 20 25 30 Leu Ser
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Arg Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser
Leu Arg Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105 110 71011PRTArtificialSynthetic Peptide
710Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 10
71111PRTArtificialSynthetic Peptide 711Arg Ala Ser Gln Gly Ile Ser
Asn Tyr Leu Ala 1 5 10 71211PRTArtificialSynthetic Peptide 712Arg
Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala 1 5 10
7137PRTArtificialSynthetic Peptide 713Ala Ala Ser Ser Leu Gln Ser 1
5 7147PRTArtificialSynthetic Peptide 714Ala Ala Ser Thr Leu Gln Ser
1 5 7157PRTArtificialSynthetic Peptide 715Lys Ala Ser Ser Leu Glu
Ser 1 5 7165PRTArtificialSynthetic Peptide 716Ser Asn Tyr Met Ser 1
5 71716PRTArtificialSynthetic Peptide 717Val Ile Tyr Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15
71817PRTArtificialSynthetic Peptide 718Val Ile Tyr Ser Gly Gly Ser
Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
719330PRTArtificialSynthetic Polypeptide 719Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175
Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180
185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305
310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
720297DNAArtificialSynthetic Sequence 720atccagatga cccagtctcc
ttcctccctg tctgcatctg taggagacag agtcaccatc 60acttgccagg ccagtcagag
cattaacaat gagttatcct ggtatcagca gaaaccaggg 120aaagccccta
agctcctgat ctatagggca tccactctgg catctggggt cccatcaagg
180ttcagcggca gtggatctgg gacagacttc actctcacca tcagcagcct
gcagcctgat 240gattttgcaa cttattactg ccaacagggt tatagtctga
ggaacattga
taatgct 297721333DNAArtificialSynthetic Sequence 721gcctatgata
tgacccagac tccagcctcg gtgtctgcag ctgtgggagg cacagtcacc 60atcaagtgcc
aggccagtca gagcattaac aatgaattat cctggtatca gcagaaacca
120gggcagcgtc ccaagctcct gatctatagg gcatccactc tggcatctgg
ggtctcatcg 180cggttcaaag gcagtggatc tgggacagag ttcactctca
ccatcagcga cctggagtgt 240gccgatgctg ccacttacta ctgtcaacag
ggttatagtc tgaggaatat tgataatgct 300ttcggcggag ggaccgaggt
ggtggtcaaa cgt 333722648DNAArtificialSynthetic Sequence
722atccagatga cccagtctcc ttcctccctg tctgcatctg taggagacag
agtcaccatc 60acttgccagg ccagtcagag cattaacaat gagttatcct ggtatcagca
gaaaccaggg 120aaagccccta agctcctgat ctatagggca tccactctgg
catctggggt cccatcaagg 180ttcagcggca gtggatctgg gacagacttc
actctcacca tcagcagcct gcagcctgat 240gattttgcaa cttattactg
ccaacagggt tatagtctga ggaacattga taatgctttc 300ggcggaggga
ccaaggtgga aatcaaacgt acggtggctg caccatctgt cttcatcttc
360ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct
gctgaataac 420ttctatccca gagaggccaa agtacagtgg aaggtggata
acgccctcca atcgggtaac 480tcccaggaga gtgtcacaga gcaggacagc
aaggacagca cctacagcct cagcagcacc 540ctgacgctga gcaaagcaga
ctacgagaaa cacaaagtct acgcctgcga agtcacccat 600cagggcctga
gctcgcccgt cacaaagagc ttcaacaggg gagagtgt
648723333DNAArtificialSynthetic Sequence 723gctatccaga tgacccagtc
tccttcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc aggccagtca
gagcattaac aatgagttat cctggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctatagg gcatccactc tggcatctgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagac ttcactctca ccatcagcag
cctgcagcct 240gatgattttg caacttatta ctgccaacag ggttatagtc
tgaggaacat tgataatgct 300ttcggcggag ggaccaaggt ggaaatcaaa cgt
333724327DNAArtificialSynthetic Sequence 724gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
ctccctcagt aactactacg tgacctgggt ccgtcaggct 120ccagggaagg
ggctggagtg ggtcggcatc atctatggta gtgatgaaac cgcctacgct
180acctccgcta taggccgatt caccatctcc agagacaatt ccaagaacac
cctgtatctt 240caaatgaaca gcctgagagc tgaggacact gctgtgtatt
actgtgctag agatgatagt 300agtgactggg atgcaaagtt caacttg
327725351DNAArtificialSynthetic Sequence 725cagtcgctgg aggagtccgg
gggtcgcctg gtcacgcctg ggacacccct gacactcacc 60tgcacagcct ctggattctc
cctcagtaac tactacgtga cctgggtccg ccaggctcca 120gggaaggggc
tggaatggat cggaatcatt tatggtagtg atgaaacggc ctacgcgacc
180tgggcgatag gccgattcac catctccaaa acctcgacca cggtggatct
gaaaatgacc 240agtctgacag ccgcggacac ggccacctat ttctgtgcca
gagatgatag tagtgactgg 300gatgcaaaat ttaacttgtg gggccaaggc
accctggtca ccgtctcgag c 351726224PRTArtificialSynthetic Polypeptide
726Met Glu Lys Leu Leu Cys Phe Leu Val Leu Thr Ser Leu Ser His Ala
1 5 10 15 Phe Gly Gln Thr Asp Met Ser Arg Lys Ala Phe Val Phe Pro
Lys Glu 20 25 30 Ser Asp Thr Ser Tyr Val Ser Leu Lys Ala Pro Leu
Thr Lys Pro Leu 35 40 45 Lys Ala Phe Thr Val Cys Leu His Phe Tyr
Thr Glu Leu Ser Ser Thr 50 55 60 Arg Gly Tyr Ser Ile Phe Ser Tyr
Ala Thr Lys Arg Gln Asp Asn Glu 65 70 75 80 Ile Leu Ile Phe Trp Ser
Lys Asp Ile Gly Tyr Ser Phe Thr Val Gly 85 90 95 Gly Ser Glu Ile
Leu Phe Glu Val Pro Glu Val Thr Val Ala Pro Val 100 105 110 His Ile
Cys Thr Ser Trp Glu Ser Ala Ser Gly Ile Val Glu Phe Trp 115 120 125
Val Asp Gly Lys Pro Arg Val Arg Lys Ser Leu Lys Lys Gly Tyr Thr 130
135 140 Val Gly Ala Glu Ala Ser Ile Ile Leu Gly Gln Glu Gln Asp Ser
Phe 145 150 155 160 Gly Gly Asn Phe Glu Gly Ser Gln Ser Leu Val Gly
Asp Ile Gly Asn 165 170 175 Val Asn Met Trp Asp Phe Val Leu Ser Pro
Asp Glu Ile Asn Thr Ile 180 185 190 Tyr Leu Gly Gly Pro Phe Ser Pro
Asn Val Leu Asn Trp Arg Ala Leu 195 200 205 Lys Tyr Glu Val Gln Gly
Glu Val Phe Thr Lys Pro Gln Leu Trp Pro 210 215 220
727468PRTArtificialSynthetic Polypeptide 727Met Leu Ala Val Gly Cys
Ala Leu Leu Ala Ala Leu Leu Ala Ala Pro 1 5 10 15 Gly Ala Ala Leu
Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg 20 25 30 Gly Val
Leu Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys Pro 35 40 45
Gly Val Glu Pro Glu Asp Asn Ala Thr Val His Trp Val Leu Arg Lys 50
55 60 Pro Ala Ala Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg
Arg 65 70 75 80 Leu Leu Leu Arg Ser Val Gln Leu His Asp Ser Gly Asn
Tyr Ser Cys 85 90 95 Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val His
Leu Leu Val Asp Val 100 105 110 Pro Pro Glu Glu Pro Gln Leu Ser Cys
Phe Arg Lys Ser Pro Leu Ser 115 120 125 Asn Val Val Cys Glu Trp Gly
Pro Arg Ser Thr Pro Ser Leu Thr Thr 130 135 140 Lys Ala Val Leu Leu
Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp 145 150 155 160 Phe Gln
Glu Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys 165 170 175
Gln Leu Ala Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met 180
185 190 Cys Val Ala Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr
Phe 195 200 205 Gln Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn
Ile Thr Val 210 215 220 Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser
Val Thr Trp Gln Asp 225 230 235 240 Pro His Ser Trp Asn Ser Ser Phe
Tyr Arg Leu Arg Phe Glu Leu Arg 245 250 255 Tyr Arg Ala Glu Arg Ser
Lys Thr Phe Thr Thr Trp Met Val Lys Asp 260 265 270 Leu Gln His His
Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His 275 280 285 Val Val
Gln Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser 290 295 300
Glu Trp Ser Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser 305
310 315 320 Pro Pro Ala Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu
Thr Thr 325 330 335 Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg Asp Ser
Ala Asn Ala Thr 340 345 350 Ser Leu Pro Val Gln Asp Ser Ser Ser Val
Pro Leu Pro Thr Phe Leu 355 360 365 Val Ala Gly Gly Ser Leu Ala Phe
Gly Thr Leu Leu Cys Ile Ala Ile 370 375 380 Val Leu Arg Phe Lys Lys
Thr Trp Lys Leu Arg Ala Leu Lys Glu Gly 385 390 395 400 Lys Thr Ser
Met His Pro Pro Tyr Ser Leu Gly Gln Leu Val Pro Glu 405 410 415 Arg
Pro Arg Pro Thr Pro Val Leu Val Pro Leu Ile Ser Pro Pro Val 420 425
430 Ser Pro Ser Ser Leu Gly Ser Asp Asn Thr Ser Ser His Asn Arg Pro
435 440 445 Asp Ala Arg Asp Pro Arg Ser Pro Tyr Asp Ile Ser Asn Thr
Asp Tyr 450 455 460 Phe Phe Pro Arg 465
728918PRTArtificialSynthetic Polypeptide 728Met Leu Thr Leu Gln Thr
Trp Val Val Gln Ala Leu Phe Ile Phe Leu 1 5 10 15 Thr Thr Glu Ser
Thr Gly Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser 20 25 30 Pro Glu
Ser Pro Val Val Gln Leu His Ser Asn Phe Thr Ala Val Cys 35 40 45
Val Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr 50
55 60 Ile Val Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr
Thr 65 70 75 80 Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp
Ile Ala Ser 85 90 95 Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr
Phe Gly Gln Leu Glu 100 105 110 Gln Asn Val Tyr Gly Ile Thr Ile Ile
Ser Gly Leu Pro Pro Glu Lys 115 120 125 Pro Lys Asn Leu Ser Cys Ile
Val Asn Glu Gly Lys Lys Met Arg Cys 130 135 140 Glu Trp Asp Gly Gly
Arg Glu Thr His Leu Glu Thr Asn Phe Thr Leu 145 150 155 160 Lys Ser
Glu Trp Ala Thr His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175
Asp Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180
185 190 Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val
Thr 195 200 205 Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys
Pro Asn Pro 210 215 220 Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu
Leu Ser Ser Ile Leu 225 230 235 240 Lys Leu Thr Trp Thr Asn Pro Ser
Ile Lys Ser Val Ile Ile Leu Lys 245 250 255 Tyr Asn Ile Gln Tyr Arg
Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile 260 265 270 Pro Pro Glu Asp
Thr Ala Ser Thr Arg Ser Ser Phe Thr Val Gln Asp 275 280 285 Leu Lys
Pro Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys Met Lys Glu 290 295 300
Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile 305
310 315 320 Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser Phe Trp Tyr
Lys Ile 325 330 335 Asp Pro Ser His Thr Gln Gly Tyr Arg Thr Val Gln
Leu Val Trp Lys 340 345 350 Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys
Ile Leu Asp Tyr Glu Val 355 360 365 Thr Leu Thr Arg Trp Lys Ser His
Leu Gln Asn Tyr Thr Val Asn Ala 370 375 380 Thr Lys Leu Thr Val Asn
Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu 385 390 395 400 Thr Val Arg
Asn Leu Val Gly Lys Ser Asp Ala Ala Val Leu Thr Ile 405 410 415 Pro
Ala Cys Asp Phe Gln Ala Thr His Pro Val Met Asp Leu Lys Ala 420 425
430 Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu
435 440 445 Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser Asp
Lys Ala 450 455 460 Pro Cys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr
Val His Arg Thr 465 470 475 480 Tyr Leu Arg Gly Asn Leu Ala Glu Ser
Lys Cys Tyr Leu Ile Thr Val 485 490 495 Thr Pro Val Tyr Ala Asp Gly
Pro Gly Ser Pro Glu Ser Ile Lys Ala 500 505 510 Tyr Leu Lys Gln Ala
Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys 515 520 525 Lys Val Gly
Lys Asn Glu Ala Val Leu Glu Trp Asp Gln Leu Pro Val 530 535 540 Asp
Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr 545 550
555 560 Ile Ile Gly Asn Glu Thr Ala Val Asn Val Asp Ser Ser His Thr
Glu 565 570 575 Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met
Val Arg Met 580 585 590 Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly
Pro Glu Phe Thr Phe 595 600 605 Thr Thr Pro Lys Phe Ala Gln Gly Glu
Ile Glu Ala Ile Val Val Pro 610 615 620 Val Cys Leu Ala Phe Leu Leu
Thr Thr Leu Leu Gly Val Leu Phe Cys 625 630 635 640 Phe Asn Lys Arg
Asp Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro 645 650 655 Asp Pro
Ser Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro 660 665 670
Arg His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe 675
680 685 Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro
Phe 690 695 700 Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu
Lys Ile Asn 705 710 715 720 Thr Glu Gly His Ser Ser Gly Ile Gly Gly
Ser Ser Cys Met Ser Ser 725 730 735 Ser Arg Pro Ser Ile Ser Ser Ser
Asp Glu Asn Glu Ser Ser Gln Asn 740 745 750 Thr Ser Ser Thr Val Gln
Tyr Ser Thr Val Val His Ser Gly Tyr Arg 755 760 765 His Gln Val Pro
Ser Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln 770 775 780 Pro Leu
Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp 785 790 795
800 His Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys
805 810 815 Gln Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His
Phe Glu 820 825 830 Arg Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp
Phe Val Arg Leu 835 840 845 Lys Gln Gln Ile Ser Asp His Ile Ser Gln
Ser Cys Gly Ser Gly Gln 850 855 860 Met Lys Met Phe Gln Glu Val Ser
Ala Ala Asp Ala Phe Gly Pro Gly 865 870 875 880 Thr Glu Gly Gln Val
Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala 885 890 895 Thr Asp Glu
Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln 900 905 910 Gly
Gly Tyr Met Pro Gln 915 72932DNAArtificialSynthetic Sequence
729agcgcttatt ccgctatcca gatgacccag tc
3273022DNAArtificialSynthetic Sequence 730cgtacgtttg atttccacct tg
2273132DNAArtificialSynthetic Sequence 731agcgcttatt ccgaggtgca
gctggtggag tc 3273220DNAArtificialSynthetic Sequence 732ctcgagacgg
tgacgagggt 20733111PRTArtificialSynthetic Polypeptide 733Ala Tyr
Asp Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Thr Ile Tyr Ser Trp 20
25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
Ile 35 40 45 Tyr Gln Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ala Gly Thr Glu Tyr Thr Leu Thr Ile
Ser Gly Val Gln Cys 65 70 75 80 Asp Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Gly Tyr Ser Gly Ser Asn 85 90 95 Val Asp Asn Val Phe Gly Gly
Gly Thr Glu Val Val Val Lys Arg 100 105 110
73499PRTArtificialSynthetic Polypeptide 734Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Phe Gly Gly Gly Thr
Lys Val Glu 85 90 95 Ile Lys Arg 73588PRTArtificialSynthetic
Polypeptide 735Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala
Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys 85
73688PRTArtificialSynthetic Polypeptide 736Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys 85
737111PRTArtificialSynthetic Polypeptide 737Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Glu Thr Ile Tyr Ser Trp 20 25 30 Leu Ser
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Gln Ala Ser Asp Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser
Gly Ser Asn 85 90 95 Val Asp Asn Val Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 100 105 110 738118PRTArtificialSynthetic
Polypeptide 738Gln Glu Gln Leu Lys Glu Ser Gly Gly Arg Leu Val Thr
Pro Gly Thr 1 5 10 15 Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe
Ser Leu Asn Asp His 20 25 30 Ala Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Phe Ile Asn Ser Gly Gly
Ser Ala Arg Tyr Ala Ser Trp Ala Glu 50 55 60 Gly Arg Phe Thr Ile
Ser Arg Thr Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Thr Ser Leu
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Val Arg Gly 85 90 95 Gly
Ala Val Trp Ser Ile His Ser Phe Asp Pro Trp Gly Pro Gly Thr 100 105
110 Leu Val Thr Val Ser Ser 115 739109PRTArtificialSynthetic
Polypeptide 739Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ala Ile Ser Ser Asn Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105
74097PRTArtificialSynthetic Polypeptide 740Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg 74197PRTArtificialSynthetic Polypeptide
741Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser
Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg
742120PRTArtificialSynthetic Polypeptide 742Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ser Ala Ser Gly Phe Ser Leu Asn Asp His 20 25 30 Ala Met
Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45
Gly Phe Ile Asn Ser Gly Gly Ser Ala Arg Tyr Ala Ser Ser Ala Glu 50
55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg Gly Gly Ala Val Trp Ser Ile His Ser Phe
Asp Pro Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115
120 7434PRTArtificialSynthetic Peptide 743Phe Gly Gly Gly 1
7444PRTArtificialSynthetic Peptide 744Val Val Lys Arg 1
74511PRTArtificialSynthetic Peptide 745Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg 1 5 10 7464PRTArtificialSynthetic Peptide 746Trp
Gly Xaa Gly 1 7474PRTArtificialSynthetic Peptide 747Thr Val Ser Ser
1 74811PRTArtificialSynthetic Peptide 748Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 1 5 10
* * * * *